Kidney-specific tumor vaccine directed against kidney tumor antigen G-250

ABSTRACT

This invention provides an anti-cancer immunogenic agent(s) (e.g. vaccines) that elicit an immune response specifically directed against renal cell cancers expressing a G250 antigenic marker. Preferred immunogenic agents comprise a chimeric molecule comprising a kidney cancer specific antigen (G250) attached to a granulocyte-macrophage colony stimulating factor (GM-CSF). The agents are useful in a wide variety of treatment modalities including, but not limited to protein vaccination, DNA vaccination, and adoptive immunotherapy.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and benefit of U.S.provisional applications U.S. Ser. No. 60/182,429, filed on Feb. 14,2000, and U.S. Ser. No. 60/182,636, filed on Feb. 15, 2000, both ofwhich are incorporated herein by reference in their entirety for allpurposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

[0002] [Not Applicable]

FIELD OF THE INVENTION

[0003] This invention relates to the field of oncology. In particularthis invention provides novel vaccines for use in the treatment of renalcell cancers.

BACKGROUND OF THE INVENTION

[0004] Renal cell carcinoma (RCC), also often identified as renalcancer, “hypemephroma”, or adenocarcinoma of the kidney accounts forabout 85 percent of all primary renal neoplasms. Approximately 25,000new cases are diagnosed annually with 10,000 deaths in the UnitedStates. Unfortunately, the prognosis of patients with recurrent ormetastatic renal cell carcinoma remains poor. Chemotherapy andradiotherapy have little or no activity in this disease and there is nostandard chemotherapeutic, hormonal, or immunologic program forrecurrent or metastatic renal cancer.

[0005] Commonly employed chemotherapy programs include the use ofvinblastine sulfate, with or without the use of nitrosoureas.Interferons have been used with very limited success. Interleukin 2(Aldesleukin) is approved for treatment of selected patients withmetastatic renal cell carcinoma. An overall response rate of 15 percenthas been noted in 255 patients, but this has been accompanied by bothsevere adverse reactions an a few treatment-related deaths. Othertreatment options for patients with advanced disease are, at best,investigational.

SUMMARY OF THE INVENTION

[0006] This invention provides a novel approach to the treatment ofrenal cell carcinomas. In particular this invention pertains to thediscovery that a chimeric molecule comprising a granulocyte macrophagecolony stimulating factor (GM-CSF) attached to a G250 kidney cancerspecific antigen provides a highly effective “vaccine” that raises animmune response directed against renal cell cancers. The chimericmolecule can be used as a traditional vaccine or in adoptiveimmunotherapeutic applications. Nucleic acids encoding a GM-CSF-G250fusion protein can be used as naked DNA vaccines or to transfect cell inan adoptive immunotherapeutic treatment regimen.

[0007] Thus in one embodiment, this invention provides a constructcomprising a G250 kidney cancer specific antigen attached to agranulocyte macrophage colony stimulating factor (GM-CSF). The GM-CSF ispreferably a human GM-CSF, or a biologically active fragment and/ormutant thereof. Similarly the G250 antigen is a preferably a human G250antigen. In particularly preferred embodiments the G250 antigen iscovalently attached to the GM-CSF (directly or through a linker).Preferred linkers are encoded by the nucleotide sequence gcggcg. In aparticularly preferred embodiment the G250 antigen and the GM-CSF arecomponents of a fusion protein (chemically constructed or recombinantlyexpressed. In such fusion proteins, the G250 antigen and the GM-CSF aredirectly joined, or more preferably, joined by a peptide linker rangingin length from 2 to about 50, more preferably from about 2 to about 20,and most preferably from about 2 to about 10 amino acids. One preferredpeptide linker is -Arg-Arg-. A particularly preferred has the sequenceof SEQ ID NO: 1 (excluding the His₆ tag).

[0008] In another embodiment this invention provides a compositioncomprising the chimeric molecules described herein and apharmaceutically acceptable diluent or excipient.

[0009] This invention also provides a nucleic acid (e.g. a DNA or anRNA) encoding a fusion protein comprising a G250 kidney cancer specificantigen attached to a granulocyte macrophage colony stimulating factor(GM-CSF). The G250 is preferably a human G250 (or an antigenic fragmentor cancer-specific epitope thereof). Similarly the GM-CSF is apreferably a human GM-CSF or a biologically active fragment thereof. Inone preferred embodiment the nucleic acid encodes a fusion protein wherethe G250 antigen and the GM-CSF are directly joined, or more preferably,joined by a peptide linker ranging in length from 2 to about 50, morepreferably from about 2 to about 20, and most preferably from about 2 toabout 10 amino acids. In certain embodiments, the nucleic acid maypreferably encode a linker that is -Arg-Arg-. One preferred nucleic acidis the nucleic acid of SEQ ID NO: 2. In some preferred embodiments, thenucleic acid is a nucleic acid that encodes the polypeptide of SEQ IDNO: 1. The nucleic acid is preferably in an expression cassette and incertain embodiments, the nucleic acid is present in a vector (e.g. abaculoviral vector).

[0010] This invention also provides a host cell transfected with one ormore of the nucleic acids described herein. The host cell is preferablya eukaryotic cell, and most preferably an insect cell.

[0011] This invention also provides methods of producing an anti-tumorvaccine. The methods preferably involve culturing a cell transfectedwith a nucleic acid encoding a chimeric GM-CSF-G250 chimeric moleculeunder conditions where the nucleic expresses a G250-GM-CSF fusionprotein and recovering said fusion protein. Again the cell is preferablya eukaryotic cell, more preferably an insect (e.g. an SF9) cell.

[0012] In another embodiment, this invention provides methods ofinducing an immune response against the G250 kidney-specific antigen,and/or a cell displaying the G250 kidney-specific antigen, and/or anycancer cell that expresses a G250 antigen, and/or an antigencross-reactive with a G250 antigen. The methods involve activating acell of the immune system with a construct comprising a kidney cancerspecific antigen (G250) attached to a granulocyte macrophage colonystimulating factor (GM-CSF) whereby the activating provides an immuneresponse directed against the G250 antigen. In some embodiments, theactivating comprises contacting an antigen presenting cell (e.g.monocyte, or dendritic cell) with the construct (chimeric molecule). Incertain embodiments, the activated cell is a cytotoxic T-lymphocyte(CTL), or a tumor infiltrating lymphocyte, etc. The activating can alsoinvolve contacting a peripheral blood lymphocyte (PBL) or a tumorinfiltrating lymphocyte (TIL) with the construct. The contacting cantake place in vivo, or ex vivo (e.g., in vitro). In various embodiments,the activating comprises loading an antigen presenting cell (APC) with apolypeptide comprising a G250. The activation can also comprisetransfecting a cell (e.g., a PBL, an APC, a TIL, a renal cell carcinomatumor cell, etc.) with a nucleic acid encoding a GM-CSF-G250 fusionprotein. The method may further comprise infusing cells (e.g. cytotoxicT lymphocytes) back into the mammal.

[0013] In still another embodiment this invention provides a method ofinhibiting the proliferation or growth of a transformed (e.g.neoplastic) kidney cell. The method involves activating a cell of theimmune system with a construct comprising a kidney cancer specificantigen (G250) attached to a granulocyte macrophage colony stimulatingfactor (GM-CSF) whereby the activating provides an immune responsedirected against the G250 antigen and the immune response inhibits thegrowth or proliferation of a transformed kidney cell. In preferredembodiments, the transformed kidney cell is a renal cell carcinoma cell(e.g. in a solid tumor, a disperse tumor, or a metastatic cell). Theactivating can comprise contacting an antigen presenting cell (e.g. adendritic cell) with the construct. The activated cell can include, butis not limited to a cytotoxic T-lymphocyte (CTL) a tumor infiltratinglymphocyte (TIL), etc. In certain embodiments, the activating comprisesinjecting (or otherwise administering) to a mammal one or more of thefollowing: a polypeptide comprising a GM-CSF-G250 fusion protein;dendritic cells pulsed with a GM-CSF-G250 fusion protein; a gene therapyconstruct (e.g. adenovirus, gutless-adenovirus, retrovirus, lentivirus,adeno-associated virus, vaccinia virus, etc) comprising a nucleic acidencoding a GM-CSF-G250 fusion protein, a dendritic expressing aGM-CSF-G250 fusion protein, a tumor cell (e.g. RCC) expressing aGM-CSF-G250 fusion protein, a fibroblast expressing a GM-CSF-G250 fusionprotein, a GM-CSF-G250 naked DNA, a transfection reagent (e.g. cationiclipid, dendrimer, liposome, etc. containing or complexed with a nucleicacid encoding a GM-CSF-G250 polypeptide. In a particularly preferredembodiment, activating comprises activating isolated dendriticcells/PMBCs. In another embodiment, the activating comprises contacting(in vivo or ex vivo) a peripheral blood lymphocyte (PBL) or a tumorinfiltrating lymphocyte (TIL) with said construct. The peripheral bloodcells and/or dendritic cells and/or monocytes are preferably infusedinto the subject.

[0014] This invention also provides a method of inhibiting theproliferation or growth of a transformed renal cell that bears a G250antigen. The method involves removing an immune cell from a mammalianhost; activating the immune cell by contacting the cell with a proteincomprising a renal cell carcinoma specific antigen (G250) attached to agranulocyte macrophage colony stimulating factor (GM-CSF) or a fragmentthereof, optionally expanding the activated cell; and infusing theactivated cell into an organism containing a transformed renal cellbearing a G250 antigen. In certain embodiments, the activating comprisescontacting the cell with one or more of the following: a polypeptidecomprising a GM-CSF-G250 fusion protein; dendritic cells pulsed with aGM-CSF-G250 fusion protein; a gene therapy construct (e.g. adenovirus,gutless-adenovirus, retrovirus, lantivirus, adeno-associated virus,vaccinia virus, etc) comprising a nucleic acid encoding a GM-CSF-G250fusion protein, a dendritic expressing a GM-CSF-G250 fusion protein, atumor cell (e.g. RCC) expressing a GM-CSF-G250 fusion protein, afibroblast expressing a GM-CSF-G250 fusion protein, a GM-CSF-G250 nakedDNA, a transfection reagent (e.g. cationic lipid, dendrimer, liposome,etc. containing or complexed with a nucleic acid encoding a GM-CSF-G250polypeptide. In a particularly preferred embodiment, activatingcomprises activating isolated dendritic cells/PMBCs. In anotherembodiment, the activating comprises contacting (in vivo or ex vivo) aperipheral blood lymphocyte (PBL) or a tumor infiltrating lymphocyte(TIL) with said construct. The peripheral blood cells and/or dendriticcells and/or monocytes are preferably infused into the subject. Theremoving may comprise isolating and culturing peripheral bloodlymphocytes and/or monocytes, and/or dendritic cells from the mammalianhost. The infusing may involve infusing the cultured cells or activatedcells produced using the cultured cells into the host from which theimmune cell was removed.

[0015] In still another embodiment, this invention provides a method oftreating an individual having a renal cell cancer. The method involvessensitizing antigen presenting cells (e.g., PBMCs, dendritic cells,etc.) in vitro with a sensitizing-effective amount of a chimeric fusionprotein comprising a renal cell carcinoma specific antigen (G250)attached to a granulocyte macrophage colony stimulating factor (GM-CSF);and administering to an individual having said renal cell cancer ormetastasis a therapeutically effective amount of the sensitized antigenpresenting cells. In particularly preferred embodiments, the antigenpresenting cells are autologous to the individual or allogenic withmatched MHC. In certain embodiments, the sensitizing involves contactingperipheral blood lymphocytes or monocytes or dendritic cells withG250-GM-CSF fusion protein. In certain embodiments, the sensitizinginvolves contacting PBL, TIL, monocyte, dendritic cell with aG250-GM-CSF polypeptide and/or transfecting dendritic cell, APC, RCC,fibroblasts, with a nucleic acid encoding the chimeric fusion protein.

[0016] Definitions

[0017] The term “G250-GM-CSF” refers to a chimeric molecule comprising aG250 renal cell tumor antigen attached to a granulocyte-macrophagecolony stimulating factor. The attachment may be a chemical conjugation(direct or through a linker) or the chimeric molecule can be a fusionprotein (recombinantly expressed or assembled by condensation of the twosubject molecules). The notation “G250-GM-CSF” encompasses embodimentswhere the G250 and the GM-CSF are attached terminally or to an internalsite and contemplates attachment of the G250 molecule to either theamino or carboxyl terminus of the GM-CSF. In addition, the term myencompass chimeric molecules comprising fragments or mutants of G250where the G250 fragments retain the epitope recognized by antibodiesthat specifically target renal cell carcinomas bearing the G250 antigen.Similarly, the term my encompass chimeric molecules comprising fragmentsor mutants of GM-CSF where the GM-CSF retain the biological activity ofnative GM-CSF (e.g. are recognized by receptors that recognize nativeGM-CSF and/or show similar mitogenic activity, etc.)

[0018] The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers. The term also includes variants on the traditional peptidelinkage joining the amino acids making up the polypeptide.

[0019] The terms “nucleic acid” or “oligonucleotide” or grammaticalequivalents herein refer to at least two nucleotides covalently linkedtogether. A nucleic acid of the present invention is preferablysingle-stranded or double stranded and will generally containphosphodiester bonds, although in some cases, as outlined below, nucleicacid analogs are included that may have alternate backbones, comprising,for example, phosphoramide (Beaucage et al. (1993) Tetrahedron49(10):1925) and references therein; Letsinger (1970) J. Org. Chem.35:3800; Sprinzl et al. (1977) Eur. J. Biochem. 81: 579; Letsinger etal. (1986) Nucl. Acids Res. 14: 3487; Sawai et al. (1984) Chem. Lett.805, Letsinger et al. (1988) J. Am. Chem. Soc. 110: 4470; and Pauwels etal. (1986) Chemica Scripta 26: 1419), phosphorothioate (Mag et al.(1991) Nucleic Acids Res. 19:1437; and U.S. Pat. No. 5,644,048),phosphorodithioate (Briu et al. (1989) J. Am. Chem. Soc. 111 :2321,O-methylphophoroamidite linkages (see Eckstein, Oligonucleotides andAnalogues: A Practical Approach, Oxford University Press), and peptidenucleic acid backbones and linkages (see Egholm (1992) J. Am. Chem. Soc.114:1895; Meier et al. (1992) Chem. Int. Ed. Engl. 31: 1008; Nielsen(1993) Nature, 365: 566; Carlsson et al. (1996) Nature 380: 207). Otheranalog nucleic acids include those with positive backbones (Denpcy etal. (1995) Proc. Natl. Acad. Sci. USA 92: 6097; non-ionic backbones(U.S. Pat. Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141 and4,469,863; Angew. (1991) Chem. Intl. Ed. English 30: 423; Letsinger etal. (1988) J. Am. Chem. Soc. 110:4470; Letsinger et al. (1994)Nucleoside & Nucleotide 13:1597; Chapters 2 and 3, ASC Symposium Series580, “Carbohydrate Modifications in Antisense Research”, Ed. Y. S.Sanghui and P. Dan Cook; Mesmaeker et al. (1994), Bioorganic & MedicinalChem. Lett. 4: 395; Jeffs et al. (1994) J. Biomolecular NMR 34:17;Tetrahedron Lett. 37:743 (1996)) and non-ribose backbones, includingthose described in U.S. Pat. Nos. 5,235,033 and 5,034,506, and Chapters6 and 7, ASC Symposium Series 580, Carbohydrate Modifications inAntisense Research, Ed. Y. S. Sanghui and P. Dan Cook. Nucleic acidscontaining one or more carbocyclic sugars are also included within thedefinition of nucleic acids (see Jenkins et al. (1995), Chem. Soc. Rev.pp169-176). Several nucleic acid analogs are described in Rawls, C & ENews Jun. 2, 1997 page 35. These modifications of the ribose-phosphatebackbone may be done to facilitate the addition of additional moietiessuch as labels, or to increase the stability and half-life of suchmolecules in physiological environments.

[0020] The term “immune cell” refers to a cell that is capable ofparticipating, directly or indirectly, in an immune response. Immunecells include, but are not limited to T-cells, B-cells, dendritic cells,cytotoxic T-cells, tumor infiltrating lymphocytes, etc.

[0021] As used herein, the term “activating” (e.g. as in activating acell or activating an immune response) includes direct activation as bycontact with the construct or by indirect activation as by contact withthe construct or antigenic fragment via an antigen presenting cell (e.g.a dendritic cell).

[0022] A “fusion protein” refers to a polypeptide formed by the joiningof two or more polypeptides through a peptide bond formed between theamino terminus of one polypeptide and the carboxyl terminus of anotherpolypeptide. The fusion protein may be formed by the chemical couplingof the constituent polypeptides, or it may be expressed as a singlepolypeptide from nucleic acid sequence encoding the single contiguousfusion protein. A single chain fusion protein is a fusion protein havinga single contiguous polypeptide backbone.

[0023] A “spacer” or “linker” as used in reference to a fusion proteinrefers to a peptide that joins the proteins comprising a fusion protein.Generally a spacer has no specific biological activity other than tojoin the proteins or to preserve some minimum distance or other spatialrelationship between them. However, the constituent amino acids of aspacer may be selected to influence some property of the molecule suchas the folding, net charge, or hydrophobicity of the molecule.

[0024] A “spacer” or “linker” as used in reference to a chemicallyconjugated chimeric molecule refers to any molecule that links/joins theconstituent molecules of the chemically conjugated chimeric molecule.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025]FIG. 1 illustrates a RT-PCR analysis of RCC tumor cells.

[0026]FIG. 2 illustrates FACS analysis of dendritic cells derived fromadherent PBMC cultures.

[0027]FIG. 3 illustrates upregulation of HLA antigen in dendritic cellsby GM-CSF-G250 fusion protein.

[0028]FIG. 4 illustrates cytotoxicity of bulk PMBC modulated byG250-GM-CSF fusion protein (patient 1).

[0029]FIG. 5 illustrates cytotoxicity of bulk PBMC modulated byGM-cSF/G250 fusion protein (patient 2).

[0030]FIGS. 6A, 6B and 6C illustrate the expression and purification ofGM-CSF-G250 fusion protein.

[0031]FIG. 6A shows immunohistochemical staining for G250 and GM-CSFexpression with anti-G250 and anti-GM-CSF antibodies Sf-9 cells infectedwith and without fusion gene recombinant baculovirus. Magnification,×100.

[0032]FIG. 6B shows a Western blot analysis of 6× His-tagged GM-CSF-G250fusion protein eluted from the Ni-NTA affinity column using antiGM-CSFantibody (L=loading, BT=break through, W=wash).

[0033]FIG. 6C shows a coomassive blue-stained SDS-PAGE of fusion proteineluted from Ni-NTA affinity column (lane 1) and further purified with SPSepharose/FPLC (lane 2 and lane 3).

[0034]FIGS. 7A and 7B show a comparison of functional activity ofrecombinant GM-CSF and purified GM-CSF-G250 fusion protein. GM-CSFactivity was measured using the GM-CSF dependent human cell line, TF-1.The TF-1 cells (2×104/well/ml) were cultured in the presence of seriallydiluted amount of (FIG. 7A) recombinant GM-CSF or, (FIG. 7B) purifiedGM-CSF-G250 fusion protein as indicated. After a 5-day incubation thecultures were pulsed with 0.1 mCi tritiated thymidine for an additional12 h. The cultures were then harvested and the incorporated thymidinemeasured by scintillation counting.

[0035]FIGS. 8A, 8B, and 8C show the immunomodulatory effects of fusionprotein on dendrtic cells.

[0036]FIG. 8A shows a double-color flow cytometric analysis of dendriticcells grown in GM-CSF (800 U/ml) plus IL-4 (1000 U/ml) or fusion protein(FP) plus IL-4. Cells were labeled with FITC and PE conjugatedantibodies against cell surface markers of DC, as indicated. Cells thatwere larger than lymphocytes were selectively gated and negativecontrols correspond to labeling with an isotype-matched controlantibody. This analysis is representative of 5 DC cultures.

[0037]FIG. 8B shows a flow cytometric analysis of HLA antigens of DCcultured in GM-CSF plus IL-4 or Fusion protein plus IL-4. Cells labeledwith primary antibody (HLA class I or class II) and FITC-conjugatedsecondary antibody. This analysis is representative of four different DCderived from four RCC patients.

[0038]FIG. 8C shows a double-color flow cytometric analysis of DCexpressed CD83⁺CD19⁻ that cultured in the condition as indicated. Data,means of triplicate; bars, SD. This analysis is representative of fourdifferent DC derived from four RCC patients

[0039]FIG. 9 shows a time course of cytokine mRNA expression in PBMCthat were treated with GM-CSF-G250 fusion protein (FP) (2.7 mg/107cells) for various time period as indicated and then harvested forsemi-quantitative RT-PCR analysis. The 32P-labeled PCR products wereseparated by electrophoresis through a 7% acrylamide gel. Gels weredried and subjected to autoradiography. Titrated standard was preparedfrom diluted RNA samples extracted from PBMC treated with FP for 24 h.

[0040]FIGS. 10A, 10B, 10C, and 10D show growth and cytotoxicity profilesof patient-derived PBMC stimulated with GM-CSF-G250 fusion protein.

[0041]FIG. 10A shows growth expansion of PBMC (patient #1) induced byvarious immunomodulatory strategies as indicated. Cell cultures werestimulated with FP on day 0, day 6, day 12 and day 18. Culture mediumwas changed weekly but maintained in a constant volume. Cell counts wereperformed on day 20. Expansion fold was calculated by division of finalcell counts per ml with cell counts per ml seeded on day 0 (3×105cells/ml). Data, means of triplicate; bars, SD. This analysis isrepresentative of four different PBMC cultures derived from four RCCpatients, which showed a similar growth profile.

[0042]FIG. 10B shows cytotoxicity of PBMC (patient #1) againstautologous normal kidney cells, primary tumor cells and lymph nodederived tumor cells. Cytotoxicity was determined by 18-h 51Cr-releaseassay on day 21. Killing activity was expressed as the lytic units per106 effector cells. Lytic units are defined as the number of effectorcells capable of inducing 30% lysis. Spontaneous release for tumortarget was <20% of maximal release. Data, means of triplicate; bars, SD.

[0043]FIG. 10C shows the inhibition of cytotoxicity against autologousLN tumor cells by antibodies specific to T cells and HLA antigens. Tumortarget cells or PBMC were pretreated with respective antibody asindicated prior to cytotoxicity assay. Data, means of triplicate; bars,SD.

[0044]FIG. 10D: Semi-quantitative RT-PCR analysis of G250 mRNAexpression by normal kidney, primary tumor and LN derived tumor derivedfrom patient #1.

[0045]FIGS. 11A and 11B shows fusion protein induced G250 targeted andMHC restricted T cell immunity.

[0046]FIG. 11A shows cytotoxicity of PBMC against autologous andallogenic tumor targets as indicated. PBMC cultures were pretreated withIL-4 (1000 U/ml) and FP (0.34 mg/ml) or IL-4 and GM-CSF (800 U/ml) forone week and then restimulated with IL-2 and FP or IL-2 and GM-CSFweekly. Cytotoxicity was determined by 18-h 51 Cr-release assay on day35. Cytotoxicity against autologous tumor target was measured in thepresence of isotype control antibody or antibodies specific to HLA classI, HLA class II, CD3, CD4, or CD8. Data, means of triplicate; bars, SD.

[0047]FIG. 11B shows a phenotypic analysis of FP modulated PBMC thatexpressed antitumor activity.

[0048]FIG. 12 shows a map of the vector pCEP4/GMCSF-G250 where therecombinant gene is inserted between KpnI and XhoI.

[0049]FIG. 13 digestion and electrophoresis of pCEP4/GMCSF-G250 Lane 1:pCEP4/GMCSF-G250. Lane 2: pCEP4/GMCSF-G250 digested with KpnI and XhoI.M: Molecular weight marker (1 kb PLUS DNA ladder (Gibco)

DETAILED DESCRIPTION

[0050] This invention provides a novel approach to the treatment (e.g.mitigation of symptoms) of a renal cell carcinoma or any type of cancerthat expresses G250 antigen (e.g. cervical cancer) or that expresses anantigen cross-reactive with G250. In particular this invention utilizesa chimeric molecule comprising a kidney cancer specific antigen (G250)attached to a granulocyte-macrophage colony stimulating factor (GM-CSF).Without being bound to a particular theory, it is believed that thischimeric molecule affords two modes of activity. Vaccination of patientswith advanced renal cell carcinoma using a chimeric G250-GM-CSF moleculewill result in activation of the patient's dendritic cells (DC), themost potent antigen presenting cells. The dendritic cells take upGM-CSF, e.g., via the GM-CSF receptor and the attached G250 antigen isco-transported by virtue of its attachment to the GM-CSF. The dendriticcells process the G250 antigen and present G250 peptide on HLA class Iwhich then activates G250 specific cytotoxic T cells (CD3⁺CD8⁺) whichcan then lyse G250 positive kidney cancer cells. In addition, oralternatively, the G250 peptide is presented on HLA class II cells thatactivate G250 specific T helper cells which then activate or maintainthe killing activity of CTLs.

[0051] In certain embodiments, a nucleic acid encoding a G250-GM-CSFconstruct can be administered as a “naked DNA” vaccine. In thisapproach, the organism/patient is injected, e.g. intramuscularly, with anucleic acid encoding a G250-GM-CSF fusion protein. The nucleic acid isexpressed within the organism leading to the production of a G250-GM-CSFfusion protein which then elicits an anti-renal cell carcinoma immuneresponse as described above.

[0052] In another embodiment, the chimeric G250-GM-CSF molecules can beused in adoptive immunotherapy. In this instance, the chimeric molecule(fusion protein) or a nucleic acid encoding the chimeric molecule isused to activate lymphocytes (e.g. T-cells) ex vivo. The activatedlymphocytes are optionally expanded, ex vivo, and then re-infused backinto the subject (patient) where they specifically attack and lyse G250positive tumor cells (e.g. kidney cells tumor or cervical cancer cells).

[0053] In particularly preferred embodiments, this invention utilizesone or more of the following formulations:

[0054] 1. A polypeptide comprising a GM-CSF-G250 fusion protein

[0055] 2. Dendritic, or other cells, pulsed with a polypeptidecomprising a GM-CSF-G250 fusion protein;

[0056] 3. GM-CSF-G250 encoding nucleic acids in a “gene therapy” vector(e.g. adenovirus, gutless-adenovirus, retrovirus, lantivirus,adeno-associated virus, vaccinia virus, etc.)

[0057] 4. Dendritic cells transfected with a GM-CSF-G250-encodingnucleic acid (e.g., via recombinant virus, plasmid DNA transfection, andthe like);

[0058] 5. Tumor cells (e.g. RCC cells) comprising a nucleic acidencoding a polypeptide comprising a GM-CSF-G250 fusion protein;

[0059] 7. A nucleic acid encoding a GM-CSF-G250 (e.g. “naked DNA”); and

[0060] 8. A nucleic acid encoding a polypeptide comprising a GM-CSF-G250complexed with a transfection agent (e.g., DMRIE/DOPE lipid, dendrimers,etc.)

[0061] Each of these formulations can be directly administered to anorganism (e.g. a mammal having a cancer that expresses a G250 antigen oran antigen cross-reactive to a G250 antigen) or can be used in anadoptive immunotherapy context. In the latter approach, the adoptiveimmunotherapy preferably utilizes cells derived from peripheral blood(e.g. peripheral blood lymphocytes (PBLs) or cells derived from a tumor(e.g. tumor infiltrating lymphocytes (TILs)). Administration of theformulation results in activation and propagation of G250-targetedcytotoxic T cells in PBMC or TIL cultures. Infusion of the G250-targetedCTLs into the patient results in the development and maintenance of aG250-directed immune response.

[0062] The formulations identified above can also be administereddirectly to a mammal for “in vivo” vaccination. Thus, for example,GM-CSF-G250 polypeptides or nucleic acids endoding such polypeptides canbe administered to the organism as “traditional” vaccines. The otherimmunogenic formulations identified above, however, are also highlyactive in vivo and can also be “directly” administered to an organism asa “vaccine”. Thus, for example, dendritic cells pulsed with aGM-CSF-G250 fusion protein, dendritic, or other cells, transfected witha nucleic acid encoding a GM-CSF-G250 fusion protein, gene therapyvectors encoding a GM-CSF-G250 polypeptide, can all be administered toan organism where they induce and maintain a population of G250-directedcytotoxic T cells.

[0063] It was a discovery of this invention that the G250-GM-CSFchimeric molecules e.g. when used in vivo as a vaccine or in an adoptiveimmunotherapeutic modality induce a highly vigorous immune responsespecifically directed at renal cell carcinomas. The approach results inthe death or inhibition of neoplastic renal cells whether diffuse (e.g.motile metastatic cells) or aggregated (e.g. as in a solid tumor). Thesemethods can accompany administration of other agents (e.g.immunomodulatory or cytotoxic agents, such as cytokines or drugs).

[0064] It is recognized that the methods of this invention need not showcomplete tumor elimination (e.g. a “cure”) to be of value. Even a slightdecrease in the growth rate of a tumor, and/or in the propagation ofmetastatic, or other neoplastic, cells can be clinically relevantimproving the quality and/or duration of life. Of course, given the highefficacy observed, it is expected that the methods of this invention mayoffer a significant or complete degree of remission particularly whenused in combination with other treatment modalities (e.g. surgery,chemotherapy, interleukin therapy, TGFβ or IL-10 antisense therapy,etc.).

[0065] I. G250-GMCSF Chimeric Molecules and Their Expression.

[0066] This invention utilizes a chimeric molecule comprising a G250kidney cancer-specific antigen attached to a granulocyte-macrophagecolony stimulating factor (GM-CSF). to induce a cell-mediated immuneresponse targeted to renal tumor cells. In a chimeric molecule, two ormore molecules that exist separately in their native state are joinedtogether to form a single molecule having the desired functionality ofall of its constituent molecules. In this instance, the constituentmolecules are the G250 antigen and GM-CSF respectively. The G250provides an epitope that is presented (e.g. to T-cells) resulting inactivation and expansion of those cells and the formation of cytotoxiccells (e.g. cytotoxic T lymphocytes, tumor infiltrating lymphocytes(TILs), etc.) that are direct to tumor cells bearing the G250 antigen.The GM-CSF acts both to stimulate components of the immune system (e.g.monocytes, dendritic cells, NK, PMN, PBMC, etc.) and to mediate uptakeof the associated G250 antigen by dendritic cells. In addition,particularly in adoptive immunotherapeutic modalities, the GM-CSF alsocan act as an adjuvant.

[0067] The attachment of the G250 antigen to the GM-CSF can be direct(e.g. a covalent bond) or indirect (e.g. through a linker). In addition,the G250 antigen and the GM-CSF proteins can be attached by chemicalmodification of the proteins or they can be expressed as a recombinantfusion protein. Detailed methods of producing the individual componentsand the chimeric molecule are provided below.

[0068] The G250 kidney tumor specific antigen is known to those of skillin the art (see, e.g., Oosterwijk et al. (1996) Molecularcharacterization of the Renal Cell Carcinoma-associated antigenG250,Proc. Natl. Acad. Sci., USA, 37: 461; Uemura et al., (1994) InternalImage Anti-Idiotype Antibodies Related to Renal-CellCarcinoma-Associated Antigen G250, Int. J. Cancer, 56: 609-614). TheG250 nucleic acid sequence is publicly available (see, e.g., GenBankAccession number X66839).

[0069] Similarly, the nucleic acid sequence of GM-CSF (e.g. humanGM-CSF) is well known to those of skill in the art (see, e.g., GenBankaccession no: E02287).

[0070] Using the known sequence information nucleic acids encoding G250,GM-CSF, or a chimeric G250-GM-CSF can be produced using standard methodswell known to those of skill in the art. For example, the nucleicacid(s) may be cloned, or amplified by in vitro methods, such as thepolymerase chain reaction (PCR), the ligase chain reaction (LCR), thetranscription-based amplification system (TAS), the self-sustainedsequence replication system (SSR), etc. A wide variety of cloning and invitro amplification methodologies are well known to persons of skill inthe art.

[0071] Examples of these techniques and instructions sufficient todirect persons of skill through many cloning exercises are found inBerger and Kimmel, Guide to Molecular Cloning Techniques, Methods inEnzymology 152 Academic Press, Inc., San Diego, Calif. (Berger);Sambrook et al. (1989) Molecular Cloning—A Laboratory Manual (2nd ed.)Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY,(Sambrook et al.); Current Protocols in Molecular Biology, F. M. Ausubelet al., eds., Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc., (1994Supplement) (Ausubel); Cashion et al., U.S. Pat. No. 5,017,478; andCarr, European Patent No. 0,246,864.

[0072] Examples of techniques sufficient to direct persons of skillthrough in vitro amplification methods are found in Berger, Sambrook,and Ausubel, as well as Mullis et al., (1987) U.S. Pat. No. 4,683,202;PCR Protocols A Guide to Methods and Applications (Innis et al. eds)Academic Press Inc. San Diego, Calif. (1990) (Innis); Arnheim & Levinson(Oct. 1, 1990) C&EN 36-47; The Journal Of NIH Research (1991) 3: 81-94;(Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86: 1173; Guatelli et al.(1990) Proc. Natl. Acad. Sci. USA 87, 1874; Lomell et al. (1989) J.Clin. Chem., 35: 1826; Landegren et al., (1988) Science, 241: 1077-1080;Van Brunt (1990) Biotechnology, 8: 291-294; Wu and Wallace, (1989) Gene,4: 560; and Barringer et al. (1990) Gene, 89: 117.

[0073] In addition, the cloning and expression of a GM-CSF-G250 fusiongene is described in Example 1. While the cloning and expression of arecombinant fusion protein is illustrated it will be appreciated thatthe G250 and GM-CSF proteins can be purchased and/or recombinantlyexpressed and then chemically coupled as described below.

[0074] The G250 and the GM-CSF molecules may be joined together in anyorder. Thus, the G250 can be joined to either the amino or carboxytermini of the GM-CSF. Where the molecules are chemically conjugated,they need not be joined end to end and can be attached at any convenientterminal or internal site.

[0075] The G250 and GM-CSF may be attached by any of a number of meanswell known to those of skill in the art. Typically the G250 and theGM-CSF are conjugated, either directly or through a linker (spacer).Because both molecules are polypeptides, in one embodiment, it ispreferable to recombinantly express the chimeric molecule as asingle-chain fusion protein that optionally contains a peptide spacerbetween the GM-CSF and the G250.

[0076] Means of chemically conjugating molecules are well known to thoseof skill. Polypeptides typically contain variety of functional groups;e.g., carboxylic acid (COOH) or free amine (—NH₂) groups, which areavailable for reaction with a suitable functional group on an effectormolecule to bind the effector thereto.

[0077] Alternatively, the G250 and/or the GM-CSF may be derivatized toexpose or attach additional reactive functional groups. Thederivatization may involve attachment of any of a number of linkermolecules such as those available from Pierce Chemical Company, RockfordIll.

[0078] A “linker”, as used herein, is a molecule that is used to jointhe G250 to the GM-CSF. In preferred embodiments, the linker is capableof forming covalent bonds to both the G250 and GM-CSF. Suitable linkersare well known to those of skill in the art and include, but are notlimited to, straight or branched-chain carbon linkers, heterocycliccarbon linkers, or peptide linkers. In certain embodiments, the linkersmay be joined to amino acids comprising G250 and/or GM-CSF through theirside groups (e.g., through a disulfide linkage to cysteine). However, ina preferred embodiment, the linkers will be joined to the alpha carbonamino and carboxyl groups of the terminal amino acids. The linker may bebifunctional, having one functional group reactive with a substituent onthe G250 and a different functional group reactive with a substituent onthe GM-CSF. Alternatively, the G250 and/or the GM-CSF may be derivatizedto react with a “mono-functional” linker (see, e.g., U.S. Pat. Nos.4,671,958 and 4,659,839 for procedures to generate reactive groups onpeptides).

[0079] In a particularly preferred embodiment, the chimeric molecules ofthis invention are fusion proteins. The fusion protein can be chemicallysynthesized using standard chemical peptide synthesis techniques, or,more preferably, recombinantly expressed. Where both molecules arerelatively short the chimeric molecule may be synthesized as a singlecontiguous polypeptide. Solid phase synthesis in which the C-terminalamino acid of the sequence is attached to an insoluble support followedby sequential addition of the remaining amino acids in the sequence is apreferred method for the chemical synthesis of the polypeptides of thisinvention. Techniques for solid phase synthesis are described by Baranyand Merrifield, Solid-Phase Peptide Synthesis; pp. 3-284 in ThePeptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods inPeptide Synthesis, Part A., Merrifield, et al. J. Am. Chem. Soc., 85:2149-2156 (1963), and Stewart et al., Solid Phase Peptide Synthesis, 2nded. Pierce Chem. Co., Rockford, Ill. (1984).

[0080] In a most preferred embodiment, the chimeric fusion proteins ofthe present invention are synthesized using recombinant DNA methodology.Generally this involves creating a DNA sequence that encodes the fusionprotein, placing the DNA in an expression cassette under the control ofa particular promoter, expressing the protein in a host, isolating theexpressed protein and, if required, renaturing the protein.

[0081] DNA encoding the fusion protein of this invention (GM-CSF-G250)may be prepared by any suitable method, including, for example, cloningand restriction of appropriate sequences or direct chemical synthesis bymethods such as the phosphotriester method of Narang et al Meth.Enzymol. 68: 90-99 (1979); the phosphodiester method of Brown et al.,Meth. Enzymol. 68: 109-151 (1979); the diethylphosphoramidite method ofBeaucage et al., Tetra. Lett., 22: 1859-1862 (1981); and the solidsupport method of U.S. Pat. No. 4,458,066.

[0082] Chemical synthesis produces a single stranded oligonucleotide.This may be converted into double stranded DNA by hybridization with acomplementary sequence, or by polymerization with a DNA polymerase usingthe single strand as a template. One of skill would recognize that whilechemical synthesis of DNA is limited to sequences of about 100 bases,longer sequences may be obtained by the ligation of shorter sequences.

[0083] Alternatively, subsequences may be cloned and the appropriatesubsequences cleaved using appropriate restriction enzymes. Thefragments may then be ligated to produce the desired DNA sequence.

[0084] In a preferred embodiment, DNA encoding fusion proteins of thepresent invention is using DNA amplification methods such as polymerasechain reaction (PCR). As illustrated in Examples 1 and 2. Thus, forexample, GM-CSF is amplified using primers that introduce EcoRI and NotIsites (3′ and 5′ respectively), and G250 cDNA is amplified with primersintroducing NotI and His-stop-GbL II (5′ and 3′ respectively. Theamplification products are ligated (GM-CSF-NotI-G250-His-stop-Bgl II).

[0085] The constructs illustrated in Example 1 introduce a linker(gcggcg) between the nucleic acids encoding G250 and GM-CSF. The linkersequence is used to separate GM-CSF and G250 by a distance sufficient toensure that, in a preferred embodiment, each domain properly folds intoits secondary and tertiary structures. Preferred peptide linkersequences adopt a flexible extended conformation, do not exhibit apropensity for developing an ordered secondary structure that couldinteract with the functional GM-CSF and G250 domains. Typical aminoacids in flexible protein regions include Gly, Asn and Ser. Virtuallyany permutation of amino acid sequences containing Gly, Asn and Serwould be expected to satisfy the above criteria for a linker sequence.Other near neutral amino acids, such as Thr and Ala, also may be used inthe linker sequence. Thus, amino acid sequences useful as linkers ofGM-CSF and G250, in addition to the one illustrated in Example 1,include the Gly₄SerGly₅Ser linker (SEQ ID NO:3) used in U.S. Pat. No.5,108,910 or a series of four (Ala Gly Ser) residues (SEQ ID NO:4), etc.Still other amino acid sequences that may be used as linkers aredisclosed in Maratea et al. (1985), Gene 40: 39-46; Murphy et al. (1986)Proc. Nat'l. Acad. Sci. USA 83: 8258-62; U.S. Pat. No. 4,935,233; andU.S. Pat. No. 4,751,180.

[0086] The length of the peptide linker sequence may vary withoutsignificantly affecting the biological activity of the fusion protein.In one preferred embodiment of the present invention, a peptide linkersequence length of about 2 amino acids is used to provide a suitableseparation of functional protein domains, although longer linkersequences also may be used. The linker sequence may be from 1 to 50amino acids in length. In the most preferred aspects of the presentinvention, the linker sequence is from about 1-20 amino acids in length.In the specific embodiments disclosed herein, the linker sequence isfrom about 2 to about 15 amino acids, and is advantageously from about 2to about 10 amino acids. Peptide linker sequences not necessarilyrequired in the fusion proteins of this invention.

[0087] Generally the spacer will have no specific biological activityother than to join the proteins or to preserve some minimum distance orother spatial relationship between them. However, the constituent aminoacids of the spacer may be selected to influence some property of themolecule such as the folding, net charge, or hydrophobicity.

[0088] Where it is desired to recombinantly express either the G250, theGM-CSF, or the G250-GM-CSF fusion protein, the nucleic acid sequencesencoding the desired protein are typically operably linked to suitabletranscriptional or translational regulatory elements. The regulatoryelements typically include a transcriptional promoter, an optionaloperator sequence to control transcription, a sequence encoding suitablemRNA ribosomal binding sites, and sequences that control the terminationof transcription and translation. The ability to replicate in a host,usually conferred by an origin of replication, and a selection gene tofacilitate recognition of transformants may additionally beincorporated.

[0089] The nucleic acid sequences encoding the fusion proteins may beexpressed in a variety of host cells, including E. coli and otherbacterial hosts, and eukaryotic host cells including but not limited toyeast, insect cells (e.g. SF9 cells) and various other eukaryotic cellssuch as the COS, CHO and HeLa cells lines and myeloma cell lines. Therecombinant protein gene will be operably linked to appropriateexpression control sequences for each host. For E. coli this includes apromoter such as the T7, trp, or lambda promoters, a ribosome bindingsite and preferably a transcription termination signal. For eukaryoticcells, the control sequences will include a promoter and preferably anenhancer derived from immunoglobulin genes, SV40, cytomegalovirus, etc.,and a polyadenylation sequence, and may include splice donor andacceptor sequences. In one particularly preferred embodiment theGM-CSF-G250 fusion gene is inserted into polyhedrin gene locus-basedbaculovirus transfer vector (e.g., pVL 1393, available from PharMingen)and expressed in insect cells (e.g. SF9 cells).

[0090] The plasmids of the invention can be transferred into the chosenhost cell by well-known methods such as calcium chloride transformationfor E. coli and calcium phosphate treatment or electroporation formammalian cells. Cells transformed by the plasmids can be selected byresistance to antibiotics conferred by genes contained on the plasmids,such as the amp, gpt, neo, and hyg genes.

[0091] Once expressed, the recombinant fusion proteins can be purifiedaccording to standard procedures of the art, including ammonium sulfateprecipitation, his tag capture, affinity columns, column chromatography,gel electrophoresis and the like (see, generally, R. Scopes, ProteinPurification, Springer-Verlag, N.Y. (1982), Deutscher, Methods inEnzymology Vol. 182: Guide to Protein Purification., Academic Press,Inc. N.Y. (1990)). Substantially pure compositions of at least about 90to 95% homogeneity are preferred, and 98 to 99% or more homogeneity aremost preferred for pharmaceutical uses. Once purified, partially or tohomogeneity as desired, the polypeptides may then be usedtherapeutically.

[0092] One of skill in the art would recognize that after chemicalsynthesis, biological expression, or purification, the G250, GM-CSF, orGM-CSF-G250 protein may possess a conformation substantially differentthan the native conformations of the polypeptide(s). In this case, itmay be necessary to denature and reduce the polypeptide and then tocause the polypeptide to re-fold into the preferred conformation.Methods of reducing and denaturing proteins and inducing re-folding arewell known to those of skill in the art (See, Debinski et al. (1993) J.Biol. Chem., 268: 14065-14070; Kreitman and Pastan, (1993) Bioconjug.Chem., 4: 581-585; and Buchner, et al., (1992) Anal. Biochem., 205:263-270). Debinski et al., for example, describe the denaturation andreduction of inclusion body proteins in guanidine-DTE. The protein isthen refolded in a redox buffer containing oxidized glutathione andL-arginine.

[0093] One of skill would recognize that modifications can be made tothe GM-CSF, G250, or GM-CSF-G250 proteins without diminishing theirbiological activity. Some modifications may be made to facilitate thecloning, expression, or incorporation of the constituent molecules intoa fusion protein. Such modifications are well known to those of skill inthe art and include, for example, a methionine added at the aminoterminus to provide an initiation site, or additional amino acids placedon either terminus to create conveniently located restriction sites ortermination codons. The recombinant expression of a GM-CSF-G250 fusionprotein is illustrated in Example 1.

[0094] II. In Vivo Protein Vaccination.

[0095] Immunogenic compositions (e.g. vaccines) are preferably preparedfrom the G250-GM-CSF fusion proteins of this invention. The immunogeniccompositions including vaccines may be prepared as injectables, asliquid solutions, suspensions or emulsions. The active immunogenicingredient or ingredients may be mixed with pharmaceutically acceptableexcipients which are compatible therewith. Such excipients are wellknown to those of skill in the art and include, but are not limited towater, saline, dextrose, glycerol, ethanol, and combinations thereof.The immunogenic compositions and vaccines may further contain auxiliarysubstances, such as wetting or emulsifying agents, pH buffering agents,or adjuvants to enhance the effectiveness thereof.

[0096] The immunogenic G250-GM-CSF compositions may be administeredparenterally, by injection subcutaneous, intravenous, intradermal,intratumoral, or intramuscularly injection. Alternatively, theimmunogenic compositions formed according to the present invention, maybe formulated and delivered in a manner to evoke an immune response atmucosal surfaces. Thus, the immunogenic composition may be administeredto mucosal surfaces by, for example, the nasal or oral (intragastric)routes. Alternatively, other modes of administration includingsuppositories and oral formulations may be desirable. For suppositories,binders and carriers may include, for example, polyalkalene glycols ortriglycerides. Such suppositories may be formed from mixtures containingthe active immunogenic ingredient (s) in the range of about 0.5 to about10%, preferably about 1 to 2%. Oral formulations may include normallyemployed carriers such as, pharmaceutical grades of saccharine,cellulose and magnesium carbonate. These compositions can take the formof solutions, suspensions, tablets, pills, capsules, sustained releaseformulations or powders and contain about 1 to 95% of the activeingredient(s), preferably about 20 to about 75%.

[0097] The immunogenic preparations and vaccines are administered in amanner compatible with the dosage formulation, and in such amount aswill be therapeutically effective, immunogenic and protective. Thequantity to be administered depends on the subject to be treated,including, for example, the capacity of the individual's immune systemto synthesize antibodies, and if needed, to produce a cell-mediatedimmune response. Precise amounts of active ingredient required to beadministered depend on the judgment of the practitioner. However,suitable dosage ranges are readily determinable by one skilled in theart and may be of the order of micrograms to milligrams of the activeingredient(s) per vaccination. The antigenic preparations of thisinvention can be administered by either single or multiple dosages of aneffective amount. Effective amounts of the compositions of the inventioncan vary from 0.01-1,000 μg/ml per dose, more preferably 0.1-500 μg/mlper dose, and most preferably 10-300 μg/ml per dose.

[0098] Suitable regimes for initial administration and booster doses arealso variable, but may include an initial administration followed bysubsequent booster administrations. The dosage may also depend or theroute of administration and will vary according to the size of the host.

[0099] The concentration of the active ingredient (chimeric protein) inan immunogenic composition according to the invention is in generalabout 1 to 95%.

[0100] Immunogenicity can be significantly improved if the antigens areco-administered with adjuvants. While the GM-CSF component of thechimeric molecule can, itself act as an adjuvant, other adjuvants can beused as well. Adjuvants enhance the immunogenicity of an antigen but arenot necessarily immunogenic themselves. Adjuvants may act by retainingthe antigen locally near the site of administration to produce a depoteffect facilitating a slow, sustained release of antigen to cells of theimmune system. Adjuvants can also attract cells of the immune system toan antigen depot and stimulate such cells to elicit immune responses.

[0101] Immunostimulatory agents or adjuvants have been used for manyyears to improve the host immune responses to, for example, vaccines.Intrinsic adjuvants, such as lipopolysaccharides, normally are thecomponents of the killed or attenuated bacteria used as vaccines.Extrinsic adjuvants are immunomodulators which are formulated to enhancethe host immune responses. Thus, adjuvants have been identified thatenhance the immune response to antigens delivered parenterally. Some ofthese adjuvants are toxic, however, and can cause undesirableside-effects, making them unsuitable for use in humans and many animals.Indeed, only aluminum hydroxide and aluminum phosphate (collectivelycommonly referred to as alum) are routinely used as adjuvants in humanand veterinary vaccines. The efficacy of alum in increasing antibodyresponses to diphtheria and tetanus toxoids is well established and aHBsAg vaccine has been adjuvanted with alum.

[0102] A wide range of extrinsic adjuvants can provoke potent immuneresponses to antigens. These include saponins complexed to membraneprotein antigens (immune stimulating complexes), pluronic polymers withmineral oil, killed mycobacteria in mineral oil, Freund's incompleteadjuvant, bacterial products, such as muramyl dipeptide (MDP) andlipopolysaccharide (LPS), as well as lipid A, and liposomes.

[0103] To efficiently induce humoral immune responses (HIR) andcell-mediated immunity (CMI), immunogens are often emulsified inadjuvants. Many adjuvants are toxic, inducing granulomas, acute andchronic inflammations (Freund's complete adjuvant, FCA), cytolysis(saponins and Pluronic polymers) and pyrogenicity, arthritis andanterior uveitis (LPS and MDP). Although FCA is an excellent adjuvantand widely used in research, it is not licensed for use in human orveterinary vaccines because of its toxicity.

[0104] III. In Vivo DNA Vaccination.

[0105] In some preferred embodiments, nucleic acids encoding aG250-GM-CSF fusion protein are incorporated into DNA vaccines. Theability of directly injected DNA, that encodes an antigenic protein, toelicit a protective immune response has been demonstrated in numerousexperimental systems (see, e.g., Conry et al. (1994) Cancer Res., 54:1164-1168; Cox et al. (1993) Virol, 67: 5664-5667; Davis et al. (1993)Hum. Mole. Genet., 2: 1847-1851; Sedegah et al. (1994) Proc. Natl. Acad.Sci., USA, 91: 9866-9870; Montgomery et al. (1993) DNA Cell Bio., 12:777-783; Ulmer et al. (1993) Science, 259: 1745-1749; Wang et al. (1993)Proc. Natl. Acad. Sci., USA, 90: 4156-4160; Xiang et al. (1994)Virology, 199: 132-140, etc.).

[0106] Vaccination through directly injecting DNA, that encodes anantigenic protein, to elicit a protective immune response often producesboth cell-mediated and humoral responses. Moreover, reproducible immuneresponses to DNA encoding various antigens have been reported in micethat last essentially for the lifetime of the animal (see, e.g.,Yankauckas et al. (1993) DNA Cell Biol., 12: 771-776).

[0107] As indicated above, DNA vaccines are known to those of skill inthe art (see, also U.S. Pat. Nos. 5,589,466 and 5,593,971,PCT/US90/01515, PCT/US93/02338, PCT/US93/04813 1, PCT/US94/00899, andthe priority applications cited therein. In addition to the deliveryprotocols described in those applications, alternative methods ofdelivering DNA are described in U.S. Pat. Nos. 4,945,050 and 5,036,006.

[0108] Using DNA vaccine technology, plasmid (or other vector) DNA thatincludes a sequence encoding a G250-GM-CSF fusion protein operablylinked to regulatory elements required for gene expression isadministered to individuals (e.g. human patients, non-human mammals,etc.). The cells of the individual take up the administered DNA and thecoding sequence is expressed. The antigen so produced becomes a targetagainst which an immune response is directed. In the present case, theimmune response directed against the antigen component of the chimericmolecule provides the prophylactic or therapeutic benefit to theindividual renal cell cancers.

[0109] The vaccines of this invention may be administered by a varietyof techniques including several different devices for administeringsubstances to tissue. The published literature includes several reviewarticles that describe aspects of DNA vaccine technology and cite someof the many reports of results obtained using the technology (see, e.g.,McDonnel and Askari (1996) New Engl. J. Med. 334(1): 42-45; Robinson(1995) Can. Med. Assoc. J. 152(10): 1629-1632; Fynan et al. (1995) Int.J Immunopharmac. 17(2): 79-83; Pardoll and Beckerleg (1995) Immunity 3:165-169; and Spooner et al. (1995) Gene Therapy 2: 173-180.

[0110] According to the present invention, the G250-GM-CSF codingsequence is inserted into a plasmid (or other vector) which is then usedin a vaccine composition. In preferred embodiments, the G250-GM-CSFcoding sequence is operably linked to regulatory elements required forexpression of the construct in eukaryotic cells. Regulatory elements forDNA expression include, but are not limited to a promoter and apolyadenylation signal. In addition, other elements, such as a Kozakregion, may also be included in the genetic construct. Initiation andtermination signals are regulatory elements which are often, but notnecessarily, considered part of the coding sequence. In preferredembodiments, the coding sequences of genetic constructs of thisinvention include functional initiation and termination signals.

[0111] Examples of promoters useful to practice the present invention,especially in the production of a genetic vaccine for humans, includebut are not limited to, promoters from Simian Virus 40 (SV40), MouseMammary Tumor Virus (MMTV) promoter, Human Immunodeficiency Virus (HIV)such as the HIV Long Terminal Repeat (LTR) promoter, Moloney virus, ALV,Cytomegalovirus (CMV) such as the CMV immediate early promoter, EpsteinBarr Virus (EBV), Rous Sarcoma Virus (RSV) as well as promoters fromhuman genes such as human Actin, human Myosin, human Hemoglobin, humanmuscle creatine and human metalothionein.

[0112] Examples of polyadenylation signals useful to practice thepresent invention, especially in the production of a genetic vaccine forhumans, include but are not limited to SV40 polyadenylation signals andLTR polyadenylation signals. In particular, the SV40 polyadenylationsignal which is in pCEP4 plasmid (Invitrogen, San Diego, Calif.),referred to as the SV40 polyadenylation signal, may be used.

[0113] In addition to the regulatory elements required for DNAexpression, other elements may also be included in the DNA molecule.Such additional elements include enhancers. The enhancer may be selectedfrom the group including but not limited to, human Actin, human Myosin,human Hemoglobin, human muscle creatine and viral enhancers such asthose from CMV, RSV and EBV.

[0114] The present invention relates to methods of introducing geneticmaterial into the cells of an individual in order to induce immuneresponses against renal cell cancers. The methods comprise the steps ofadministering to the tissue of said individual, DNA that includes acoding sequence for a G250-GM-CSF fusion protein operably linked toregulatory elements required for expression. The DNA can be administeredin the presence of adjuvants or other substances that have thecapability of promoting DNA uptake or recruiting immune system cells tothe site of the inoculation. It should be understood that, in preferredembodiments, the DNA transcription unit itself is expressed in the hostcell by transcription factors provided by the host cell, or provided bya DNA transcriptional unit. A DNA transcription unit can comprisenucleic acids that encode proteins that serve to stimulate the immuneresponse such as a cytokine, proteins that serve as an adjuvant andproteins that act as a receptor.

[0115] Vectors containing the nucleic acid-based vaccine of theinvention can be introduced into the desired host by methods known inthe art, e.g., transfection, electroporation, microinjection,transduction, cell fusion, DEAE dextran, calcium phosphateprecipitation, lipofection (lysosome fusion), use of a gene gun, or aDNA vector transporter (see, e.g., Wu et al (1992) J. Biol. Chem. 267:963-967; Wu and Wu (1988) J. Biol. Chem. 263: 14621-14624). The subjectcan be inoculated intramuscularly, intranasally, intraperatoneally,subcutaneously, intradermally, topically, or by a gene gun.

[0116] The subject can also be inoculated by a mucosal route. The DNAtranscription unit can be administered to a mucosal surface by a varietyof methods, including lavage, DNA-containing nose-drops, inhalants,suppositories or by microsphere encapsulated DNA. For example, the DNAtranscription unit can be administered to a respiratory mucosal surface,such as the trachea or into any surface including the tongue or mucousmembrane.

[0117] The DNA transcription units are preferably administered in amedium, i.e., an adjuvant, that acts to promote DNA uptake andexpression. Preferably, a pharmaceutically acceptable, inert medium issuitable as an adjuvant for introducing the DNA transcription unit intothe subject. One example of a suitable adjuvant is alum (alumina gel),though even a saline solution is acceptable. Other possible adjuvantsinclude organic molecules such as squalines, iscoms, organic oils andfats.

[0118] An immuno-effector can be co-expressed with the G250-GM-CSFnucleic acid of this present invention and thereby enhance the immuneresponse to the antigen. A nucleic acid encoding the immuno-effector maybe administered in a separate DNA transcription unit, operatively linkedto a suitable DNA promoter, or alternatively the immuno-effector may beincluded in a DNA transcription unit comprising a nucleic acid thatencodes the G250-GM-CSF construct that are operatively linked to one ormore DNA promoters. Other embodiments contain two or more suchimmuno-effectors operatively linked to one or more promoters. Thenucleic acid can consist of one contiguous polymer, encoding both thechimeric protein and the immuno-effector or it can consist ofindependent nucleic acid segments that individually encode the chimericmolecule and the immuno-effector respectively. In the latter case, thenucleic acid may be inserted into one vector or the independent nucleicacid segments can be placed into separate vectors. The nucleic acidencoding the immuno-effector and the chimeric molecule may be eitheroperatively linked to the same DNA promoter or operatively linked toseparate DNA promoters. Adding such an immuno-effector is known in theart. Alternatively, soluble immuno-effector proteins (cytokines,monokines, interferons, etc.) can be directly administered into thesubject in conjunction with the G250-GM-CSF DNA.

[0119] Examples of immuno-effectors include, but are not limited to,interferon-α, interferon-γ, interferon-β, interferon-θ, interferon-τ,tumor necrosis factor-α, tumor necrosis factor-β, interleukin-2,interleukin-6, interleukin-7, interleukin-12, interleukin-15, B7-1 Tcell co-stimulatory molecule, B7-2 T cell co-stimulatory molecule,immune cell adhesion molecule (ICAM)-1, T cell co-stimulatory molecule,granulocyte colony stimulatory factor, granulocyte-macrophage colonystimulatory factor, and combinations thereof.

[0120] When taken up by a cell, the genetic construct(s) may remainpresent in the cell as a functioning extrachromosomal molecule and/orintegrate into the cell's chromosomal DNA. DNA may be introduced intocells where it remains as separate genetic material, e.g., in the formof a plasmid or plasmids. Alternatively, linear DNA which can integrateinto the chromosome may be introduced into the cell. When introducingDNA into the cell, reagents which promote DNA integration intochromosomes may be added. DNA sequences which are useful to promoteintegration may also be included in the DNA molecule. Alternatively, RNAmay be administered to the cell. It is also contemplated to provide thegenetic construct as a linear minichromosome including a centromere,telomeres and an origin of replication. Gene constructs may remain partof the genetic material in attenuated live microorganisms or recombinantmicrobial vectors which live in cells. Gene constructs may be part ofgenomes of recombinant viral vaccines where the genetic material eitherintegrates into the chromosome of the cell or remains extrachromosomal.

[0121] Genetic constructs can be provided with a mammalian origin ofreplication in order to maintain the construct extrachromosomally andproduce multiple copies of the construct in the cell. Thus, for example,plasmids pCEP4 and pREP4 from Invitrogen (San Diego, Calif.) contain theEpstein Barr virus origin of replication and nuclear antigen EBNA-1coding region which produces high copy episomal replication withoutintegration.

[0122] An additional element may be added which serves as a target forcell destruction if it is desirable to eliminate cells receiving thegenetic construct for any reason. A herpes thymidine kinase (tk) gene inan expressible form can be included in the genetic construct. The druggangcyclovir can be administered to the individual and that drug willcause the selective killing of any cell producing tk, thus, providingthe means for the selective destruction of cells with the G250-GM-CSFnucleic acid construct.

[0123] In order to maximize protein production, regulatory sequences maybe selected which are well suited for gene expression in the cells intowhich the construct is administered. Moreover, codons may be selectedwhich are most efficiently transcribed in the cell. One having ordinaryskill in the art can produce DNA constructs which are functional in thecells.

[0124] The concentration of the dosage is preferably sufficient toprovide an effective immune response. The dosage of the recombinantvectors administered will depend upon the properties of the formulationemployed, e.g., its in vivo plasma half-life, the concentration of therecombinant vectors in the formulation, the administration route, thesite and rate of dosage, the clinical tolerance of the subject, and thelike, as is well within the skill of one skilled in the art. Differentdosages may be utilized in a series of inoculations; the practitionermay administer an initial inoculation and then boost with relativelysmaller doses of the recombinant vectors or other boosters.

[0125] The preferred dose range is between about 30 μg to about 1 mgDNA, and more preferably between about 50 μg to 500 μg. Lower doses maybe used as plasmid expression and inoculation are optimized. Dosages maydiffer for adults in contrast to adolescents or children. Theinoculation is preferably followed by boosters.

[0126] IV. Adoptive Immunotherapy.

[0127] Adoptive immunotherapy refers to a therapeutic approach fortreating cancer or infectious diseases in which immune cells areadministered to a host with the aim that the cells mediate eitherdirectly or indirectly specific immunity to (i.e., mount an immuneresponse directed against) tumor cells. In preferred embodiments, theimmune response results in inhibition of tumor and/or metastatic cellgrowth and/or proliferation and most preferably results in neoplasticcell death and/or resorption. The immune cells can be derived from adifferent organism/host (exogenous immune cells) or can be cellsobtained from the subject organism (autologous immune cells).

[0128] The immune cells are typically activated in vitro by a particularantigen (in this case G250), optionally expanded, and then re-infusedback into the source organism (e.g., patient). Methods of performingadoptive immunotherapy are well known to those of skill in the art (see,e.g., U.S. Pat. Nos. 5,081,029, 5,985,270, 5,830,464, 5,776,451,5,229,115, 690,915, and the like).

[0129] In preferred embodiments, this invention contemplates numerousmodalities of adoptive immunotherapy, e.g. as described above. In oneembodiment, dendritic cells (e.g. isolated from the patient orautologous dendritic cells) are pulsed with G250 or the G250-GM-CSFchimeric molecule and then injected back into the subject where theypresent and activate immune cells in vivo. In addition, oralternatively, the dentritic cells can be transfected with nucleic acidsencoding the G250-GM-CSF fusion protein and then re-introduced into apatient.

[0130] In another embodiment, modified macrophage or dendritic cell(antigen presenting cells) are pulsed with G250-GM-CSF fusion proteinsor transfected with nucleic acids encoding a G250-GM-CSF fusion protein,and then used to stimulate peripheral blood lymphocytes or TIL inculture and activate G250-targeted CTLs that are then infused into thepatient.

[0131] Similarly, fibroblasts, and other APCs, or tumor cells (e.g.RCCs) are transfected with a nucleic acid expressing a G250-GM-CSF andused to activate tumor cells or PBLs ex vivo to produce G250 directedCTLs that can then be infused into a patient.

[0132] Similarly various “transfection agents” including, but notlimited to gene therapy vectors (e.g. adenovirus, gutless-adenovirus,retrovirus, lantivirus, adeno-associated virus, vaccinia virus etc),cationic lipids, liposomes, dendrimers, and the like, containing orcomplexed with a nucleic acid encoding a G250-GM-CSF fusion protein areadministered to PBLs or to tumor cells (e.g. RCCs) ex vivo to produceG250 directed CTLs.

[0133] In one particularly preferred emobdiments, tumor cells (e.g. RCCcells) transfected to express a G250-GM-CSF protein are used to providean off-the-shelf vaccine effective against tumors expressing a G250antigen or an antigen that is cross-reactive with G250.

[0134] Using the teachings provided herein, other therapeutic modalitiesutilizing G250-GM-CSF polypeptides or G250-GM-CSF nucleic acids can bereadily developed.

[0135] As indicated above, in one embodiment the immune cells arederived from peripheral blood lymphocytes or TILs (e.g. derived fromtumors/tumor suspension). Lymphocytes used for in vitro activationinclude, but are not limited to T lymphocytes, various antigenpresenting cells (e.g. monocytes, dendritic cells, B cells, etc.) andthe like. Activation can involve contacting an antigen presenting cellwith the chimeric molecule(s) of this invention which then present theG250 antigen (or fragment thereof), e.g., on HLA class I moleculesand/or on HLA class II molecules, and/or can involve contacting a cell(e.g. T-lymphocyte) directly with the chimeric molecule. Theantigen-presenting cells (APCs), including but not limited tomacrophages, dendritic cells and B-cells, are preferably obtained byproduction in vitro from stem and progenitor cells from human peripheralblood or bone marrow as described by Inaba et al., (1992) J. Exp. Med.176:1693-1702.

[0136] Activation of immune cells can take a number of forms. Theseinclude, but are not limited to the direct addition of the chimericmolecule to peripheral blood lymphocytes (PBLs) or tumor infiltratinglymphocytes (TILs) in culture, loading of antigen presenting cells (e.g.monocytes, dendritic cells, etc.) with the chimeric molecule in culture,transfection of antigen presenting cells, or PBLs, with a nucleic acidencoding the GM-CSF-G250 chimeric fusion protein, and the like.

[0137] APC can be obtained by any of various methods known in the art.In a preferred aspect human macrophages and/or dendritic cells are used,obtained from human blood donors. By way of example but not limitation,PBLs (e.g. T-cells) can be obtained as follows:

[0138] Approximately 200 ml of heparinized venous blood is drawn byvenipuncture and PBL are isolated by Ficoll-hypaque gradientcentrifugation, yielding approximately 1 to 5×10⁸ PBL, depending uponthe lymphocyte count of the donor(s). The PBL are washed inphosphate-buffered saline and are suspended at approximately 2×10⁵/ml inRPMI 1640 medium containing 10% pooled heat-inactivated normal humanserum; this medium will be referred to as “complete medium.”

[0139] Similarly, other cells (e.g. mononuclear cells) are isolated fromperipheral blood of a patient (preferably the patient to be treated), byFicoll-Hypaque gradient centrifugation and are seeded on tissue culturedishes which are pre-coated with the patient's own serum or with otherAB+ human serum. The cells are incubated at 37° C. for 1 hr, thennon-adherent cells are removed by pipetting. To the adherent cells leftin the dish, is added cold (4° C.) 1 mM EDTA in phosphate-bufferedsaline and the dishes are left at room temperature for 15 minutes. Thecells are harvested, washed with RPMI buffer and suspended in RPMIbuffer. Increased numbers of macrophages may be obtained by incubatingat 37° C. with macrophage-colony stimulating factor (M-CSF); increasednumbers of dendritic cells may be obtained by incubating withgranulocyte-macrophage-colony stimulating factor (GM-CSF) as describedin detail by Inaba et al. (1992) J. Exp. Med. 176:1693-1702, and morepreferably by incubating with the G250-GM-CSF chimeric molecules of thisinvention and, optionally IL-4).

[0140] The cells (e.g. APCs) are sensitized by contacting/incubatingthem with the chimeric molecule. In some embodiments, sensitization maybe increased by contacting the APCs with heat shock protein(s) (hsp)noncovalently bound to the chimeric molecule. It has been demonstratedthat hsps noncovalently bound to antigenic molecules can increase APCsensitization in adoptive immunotherapeutic applications (see, e.g.,U.S. Pat. No. 5,885,270).

[0141] In one preferred embodiment, e.g. as described in the examplesherein, G250-GM-CSF fusion protein (with optional IL-4) is added intothe patients PBMC ex vivo and then cultured at 37° C. for 7 days. Theculture is re-stimulated weekly with IL-2 and fusion protein, e.g. for 4to 5 cycles until the culture shows anti-tumor activity againstautologous kidney tumor cells displaying G250. The CTLs are thenreinfused back into the patient.

[0142] For re-infusion, the cells are washed three times and resuspendedin a physiological medium preferably sterile, at a convenientconcentration (e.g., 1×10⁷/ml) for injection in a patient. The cellsuspension is then filtered, e.g., through sterile 110 mesh and put intoFenwall transfer packs. Samples of the cells are tested for the presenceof microorganisms including fungi, aerobic and anaerobic bacteria, andmycoplasma. A sample of the cells is optionally retained forimmunological testing in order to demonstrate induction of specificimmunity.

[0143] In a preferred embodiment, before use in immunotherapy, thestimulated lymphocytes are tested for cell-mediated immune reactivityagainst tumor cells bearing the G250 antigen. The PBL/TIL, followingstimulation with the chimeric molecules of this invention can beexamined with regard to cell surface expression of T and B cell markersby immunofluorescent analysis using fluorescein-conjugated monoclonalantibodies to T and B cell antigens. Expression of known T cell markers,such as the CD4 and CD8 antigens, confirms the identity of the activatedlymphocytes as T cells.

[0144] The activated cells (e.g. activated T cells) are then,optionally, tested for reactivity against G250. This could beaccomplished by any of several techniques known in the art for assayingspecific cell-mediated immunity. For example, a cytotoxicity assay,which measures the ability of the stimulated T cells to kill tumor cellsbearing the G250 antigen in vitro, may be accomplished by incubating thelymphocytes with G250-bearing tumor cells containing a marker (e.g.⁵¹Cr-labelled cells) and measuring ⁵¹Cr release upon lysis. Such assayshave been described (see, e.g., Zarling et al. (1986) J. Immunol. 136:4669). The activated PBL could also be tested for T helper cell activityby measuring their ability to proliferate, as shown by ³H-thymidineincorporation, following stimulation, and/or by measuring their abilityto produce lymphokines such as IL-2 or interferon upon stimulation, inthe absence of exogenous IL-2. Other assays of specific cell-mediatedimmunity known in the art, such as leukocyte-adherence inhibition assays(Thomson, D. M. P. (ed.), 1982, Assessment of Immune Status by theLeukocyte Adherence Inhibition Test, Academic Press, New York), may alsobe used.

[0145] Inoculation of the activated cells is preferably through systemicadministration. The cells can be administered intravenously through acentral venous catheter or into a large peripheral vein. Other methodsof administration (for example, direct infusion into an artery) arewithin the scope of the invention. Approximately 1×10⁸ cells are infusedinitially and the remainder are infused over the following severalhours. In some regimens, patients may optionally receive in addition asuitable dosage of a biological response modifier including but notlimited to the cytokines IFN-α, IFN-γ, IL-2, IL-4, IL-6, TNF or othercytokine growth factor, antisense TGFβ, antisense IL-10, and the like.Thus, in some patients, recombinant human IL-2 may be used and will beinfused intravenously every 8 hours beginning at the time of T cellinfusion. Injections of IL-2 will preferably be at doses of 10,000 to100,000 units/kg bodyweight, as previously used in cancer patients(Rosenberg et al. (1985) N. Engl. J. Med. 313:1485). The IL-2 infusionmaybe continued for several days after infusion of the activated T cellsif tolerated by the patient.

[0146] Treatment by inoculation of, e.g., activated T cells can be usedalone or in conjunction with other therapeutic regimens including butnot limited to administration of IL-2 (as described supra), otherchemotherapeutics (e.g. doxirubicin, vinblastine, vincristine, etc.),radiotherapy, surgery, and the like.

[0147] As indicated above, the cells may, optionally, be expanded inculture. This expansion can be accomplished by repeated stimulation ofthe T cells with the G250-GM-CSF construct of this invention with orwithout IL-2 or by growth in medium containing IL-2 alone. Other methodsof T cell cultivation (for example with other lymphokines, growthfactors, or other bioactive molecules) are also within the scope of theinvention. For example, antibodies or their derivative molecules whichrecognize the Tp67 or Tp44 antigens on T cells have been shown toaugment proliferation of activated T cells (Ledbetter et al. (1985) J.Immunol. 135: 2331), and maybe used during in vitro activation toincrease proliferation. Interferon has been found to augment thegeneration of cytotoxic T cells (Zarling et al. (1978) Immunol. 121:2002), and may be used during in vitro activation to augment thegeneration of cytotoxic T cells against G250 bearing cancer cells.

[0148] The description provided above details various methods forisolation, activation, and expansion of PBL. However the presentinvention provides for the use G250-GM-CSF constructs in various forms,and modifications and adaptations to the method to accommodate thesevariations. Thus modifications of various adoptive immunotherapeuticapproaches utilizing the G250-GM-CSF constructs are within the scope ofthe invention.

[0149] V. Gene Transfer for Systemic Therapy or for AdoptiveImmunotherapy.

[0150] In addition to use of the chimeric GM-CSF-G250 chimeric proteinfor activation in adoptive immunotherapy, cells, (e.g., APCs, PBLs,fibroblasts, TILs, or RCC tumor cells) can be transfected with a vectorexpressing the chimeric molecule and used for adoptive immunotherapyand/or vaccine therapy.

[0151] In one preferred embodiment, the nucleic acid(s) encoding theGM-CSF-G250 chimeric fusion proteins are cloned into gene therapyvectors that are competent to transfect cells (such as human or othermammalian cells) in vitro and/or in vivo.

[0152] Several approaches for introducing nucleic acids into cells invivo, ex vivo and in vitro have been used. These include lipid orliposome based gene delivery (WO 96/18372; WO 93/24640; Mannino andGould-Fogerite (1988) BioTechniques 6(7): 682-691; Rose U.S. Pat No.5,279,833; WO 91/06309; and Felgner et al. (1987) Proc. Natl. Acad. Sci.USA 84: 7413-7414) and replication-defective retroviral vectorsharboring a therapeutic polynucleotide sequence as part of theretroviral genome (see, e.g., Miller et al. (1990) Mol. Cell. Biol.10:4239 (1990); Kolberg (1992) J. NIH Res. 4: 43, and Cornetta et al.(1991) Hum. Gene Ther. 2: 215).

[0153] For a review of gene therapy procedures, see, e.g., Anderson,Science (1992) 256: 808-813; Nabel and Felgner (1993) TIBTECH 11:211-217; Mitani and Caskey (1993) TIBTECH 11: 162-166; Mulligan (1993)Science, 926-932; Dillon (1993) TIBTECH 11: 167-175; Miller (1992)Nature 357: 455-460; Van Brunt (1988) Biotechnology 6(10): 1149-1154;Vigne (1995) Restorative Neurology and Neuroscience 8: 35-36; Kremer andPerricaudet (1995) British Medical Bulletin 51(1) 31-44; Haddada et al.(1995) in Current Topics in Microbiology and Immunology, Doerfler andBöhm (eds) Springer-Verlag, Heidelberg Germany; and Yu et al., (1994)Gene Therapy, 1: 13-26.

[0154] Widely used retroviral vectors include those based upon murineleukemia virus (MuLV), gibbon ape leukemia virus (GaLV), SimianImmunodeficiency virus (SIV), human immunodeficiency virus (HIV),alphavirus, and combinations thereof (see, e.g., Buchscher et al. (1992)J. Virol. 66(5)2731-2739; Johann et al. (1992) J. Virol. 66(5):1635-1640 (1992); Sommerfelt et al., (1990) Virol. 176:58-59; Wilsonet al (1989) J. Virol. 63:2374-2378; Miller et al., J. Virol.65:2220-2224 (1991); Wong-Staal et al., PCT/US94/05700, and Rosenburgand Fauci (1993) in Fundamental Immunology, Third Edition Paul (ed)Raven Press, Ltd., New York and the references therein, and Yu et al.(1994) Gene Therapy, supra; U.S. Pat. No. 6,008,535, and the like).

[0155] The vectors are optionally pseudotyped to extend the host rangeof the vector to cells which are not infected by the retroviruscorresponding to the vector. For example, the vesicular stomatitis virusenvelope glycoprotein (VSV-G) has been used to constructVSV-G-pseudotyped HIV vectors which can infect hematopoietic stem cells(Naldini et al. (1996) Science 272:263, and Akkina et al. (1996) J.Virol 70:2581).

[0156] Adeno-associated virus (AAV)-based vectors are also used totransduce cells with target nucleic acids, e.g., in the in vitroproduction of nucleic acids and peptides, and in in vivo and ex vivogene therapy procedures. See, West et al. (1987) Virology 160:38-47;Carter et al. (1989) U.S. Pat. No. 4,797,368; Carter et al. WO 93/24641(1993); Kotin (1994) Human Gene Therapy 5:793-801; Muzyczka (1994) J.Clin. Invst. 94:1351 for an overview of AAV vectors. Construction ofrecombinant AAV vectors are described in a number of publications,including Lebkowski, U.S. Pat. No. 5,173,414; Tratschin et al. (1985)Mol. Cell. Biol. 5(11):3251-3260; Tratschin, et al. (1984) Mol. Cell.Biol., 4: 2072-2081; Hermonat and Muzyczka (1984) Proc. Natl. Acad. Sci.USA, 81: 6466-6470; McLaughlin et al. (1988) and Samulski et al. (1989)J. Virol., 63:03822-3828. Cell lines that can be transformed by rAAVinclude those described in Lebkowski et al. (1988) Mol. Cell. Biol.,8:3988-3996. Other suitable viral vectors include herpes virus,lentivirus, and vaccinia virus.

[0157] In addition to viral vectors, a number of non-viral transfectionmethods are available. Such methods include, but are not limited toelectroporation methods, calcium phosphate transfection, liposomes,cationic lipid complexes, water-oil emulsions, polethylene imines, anddendrimers.

[0158] Liposomes were first described in 1965 as a model of cellularmembranes and quickly were applied to the delivery of substances tocells. Liposomes entrap DNA by one of two mechanisms which has resultedin their classification as either cationic liposomes or pH-sensitiveliposomes. Cationic liposomes are positively charged liposomes whichinteract with the negatively charged DNA molecules to form a stablecomplex. Cationic liposomes typically consist of a positively chargedlipid and a co-lipid. Commonly used co-lipids include dioleoylphosphatidylethanolamine (DOPE) or dioleoyl phosphatidylcholine (DOPC).Co-lipids, also called helper lipids, are in most cases required forstabilization of liposome complex. A variety of positively charged lipidformulations are commercially available and many other are underdevelopment. Two of the most frequently cited cationic lipids arelipofectamine and lipofectin. Lipofectin is a commercially availablecationic lipid first reported by Phil Felgner in 1987 to deliver genesto cells in culture. Lipofectin is a mixture ofN-[1-(2,3-dioleyloyx)propyl]-N-N-N-trimethyl ammonia chloride (DOTMA)and DOPE.

[0159] DNA and lipofectin or lipofectamine interact spontaneously toform complexes that have a 100% loading efficiency. In other words,essentially all of the DNA is complexed with the lipid, provided enoughlipid is available. It is assumed that the negative charge of the DNAmolecule interacts with the positively charged groups of the DOTMA. Thelipid:DNA ratio and overall lipid concentrations used in forming thesecomplexes are extremely important for efficient gene transfer and varywith application. Lipofectin has been used to deliver linear DNA,plasmid DNA, and RNA to a variety of cells in culture. Shortly after itsintroduction, it was shown that lipofectin could be used to delivergenes in vivo. Following intravenous administration of lipofectin-DNAcomplexes, both the lung and liver showed marked affinity for uptake ofthese complexes and transgene expression. Injection of these complexesinto other tissues has had varying results and, for the most part, aremuch less efficient than lipofectin-mediated gene transfer into eitherthe lung or the liver.

[0160] PH-sensitive, or negatively-charged liposomes, entrap DNA ratherthan complex with it. Since both the DNA and the lipid are similarlycharged, repulsion rather than complex formation occurs. Yet, some DNAdoes manage to get entrapped within the aqueous interior of theseliposomes. In some cases, these liposomes are destabilized by low pH andhence the term pH-sensitive. To date, cationic liposomes have been muchmore efficient at gene delivery both in vivo and in vitro thanpH-sensitive liposomes. pH-sensitive liposomes have the potential to bemuch more efficient at in vivo DNA delivery than their cationiccounterparts and should be able to do so with reduced toxicity andinterference from serum protein.

[0161] In another approach dendrimers complexed to the DNA have beenused to transfect cells. Such dendrimers include, but are not limitedto, “starburst” dendrimers and various dendrimer polycations.

[0162] Dendrimer polycations are three dimensional, highly orderedoligomeric and/or polymeric compounds typically formed on a coremolecule or designated initiator by reiterative reaction sequencesadding the oligomers and/or polymers and providing an outer surface thatis positively changed. These dendrimers may be prepared as disclosed inPCT/US83/02052, and U.S. Pat. Nos. 4,507,466, 4,558,120, 4,568,737,4,587,329, 4,631,337, 4,694,064, 4,713,975, 4,737,550, 4,871,779,4,857,599.

[0163] Typically, the dendrimer polycations comprise a core moleculeupon which polymers are added. The polymers may be oligomers or polymerswhich comprise terminal groups capable of acquiring a positive charge.Suitable core molecules comprise at least two reactive residues whichcan be utilized for the binding of the core molecule to the oligomersand/or polymers. Examples of the reactive residues are hydroxyl, ester,amino, imino, imido, halide, carboxyl, carboxyhalide maleimide,dithiopyridyl, and sulfhydryl, among others. Preferred core moleculesare ammonia, tris-(2-aminoethyl)amine, lysine, omithine, pentaerythritoland ethylenediamine, among others. Combinations of these residues arealso suitable as are other reactive residues.

[0164] Oligomers and polymers suitable for the preparation of thedendrimer polycations of the invention are pharmaceutically-acceptableoligomers and/or polymers that are well accepted in the body. Examplesof these are polyamidoamines derived from the reaction of an alkyl esterof an α,β-ethylenically unsaturated carboxylic acid or anα,β-ethylenically unsaturated amide and an alkylene polyamine or apolyalkylene polyamine, among others. Preferred are methyl acrylate andethylenediamine. The polymer is preferably covalently bound to the coremolecule.

[0165] The terminal groups that may be attached to the oligomers and/orpolymers should be capable of acquiring a positive charge. Examples ofthese are azoles and primary, secondary, tertiary and quaternaryaliphatic and aromatic amines and azoles, which may be substituted withS or O, guanidinium, and combinations thereof. The terminal cationicgroups are preferably attached in a covalent manner to the oligomersand/or polymers. Preferred terminal cationic groups are amines andguanidinium. However, others may also be utilized. The terminal cationicgroups may be present in a proportion of about 10 to 100% of allterminal groups of the oligomer and/or polymer, and more preferablyabout 50 to 100%.

[0166] The dendrimer polycation may also comprise 0 to about 90%terminal reactive residues other than the cationic groups. Suitableterminal reactive residues other than the terminal cationic groups arehydroxyl, cyano, carboxyl, sulfhydryl, amide and thioether, amongothers, and combinations thereof. However others may also be utilized.

[0167] The dendrimer polycation is generally and preferablynon-covalently associated with the polynucleotide. This permits an easydisassociation or disassembling of the composition once it is deliveredinto the cell. Typical dendrimer polycation suitable for use herein havea molecular weight ranging from about 2,000 to 1,000,000 Da, and morepreferably about 5,000 to 500,000 Da. However, other molecule weightsare also suitable. Preferred dendrimer polycations have a hydrodynamicradius of about 11 to 60 Å, and more preferably about 15 to 55 Å. Othersizes, however, are also suitable. Methods for the preparation and useof dendrimers in gene therapy are well known to those of skill in theart and describe in detail, for example, in U.S. Pat. No. 5,661,025.

[0168] Where appropriate, two or more types of vectors can be usedtogether. For example, a plasmid vector may be used in conjunction withliposomes. In the case of non-viral vectors, nucleic acid may beincorporated into the non-viral vectors by any suitable means known inthe art. For plasmids, this typically involves ligating the constructinto a suitable restriction site. For vectors such as liposomes,water-oil emulsions, polyethylene amines and dendrimers, the vector andconstruct may be associated by mixing under suitable conditions known inthe art.

[0169] VI. Administration of GM-CSF-G250 with Other Agents.

[0170] In various embodiments, the GM-CSF-G250 fusion proteins, ornucleic acids encoding the GM-CSF-G250 fusion proteins can beadministered in conjunction with other agents. Such agents include, butare not limited to various chemotherapeutic agents (e.g. doxirubicin andderivatives, taxol and derivatives, vinblastine, vincristine,camptothecin derivatives, and the like, various cytokines (e.g. IL-2,IL-7, IL-12, IFN, etc.), various cytotoxins (e.g. Pseudomonas exotoxinand derivatives, diphtheria toxin and derivatives, ricin andderivatives, abrin and derivatives, thymidine kinase and derivatives),antisense molecules (e.g. antisense IL-10, TGF-β, etc.), antibodiesagainst various growth factors/receptors (e.g. anti-VEGF, anti-EGFR,anti-IL-8, anti-FGF etc.), and the like. The methods of this inventioncan also be used as a adjunct to surgery, and/or radiotherapy.

[0171] VII. Kits.

[0172] Kits of the invention are provided that includematerials/reagents useful for vaccination using a polypeptide antigen(GM-CSF-G250 polypeptide) and/or DNA vaccination, and/or adoptiveimmunotherapy. Kits optimized for GM-CSF-G250 polypeptide vaccinationpreferably comprise a container containing a GM-CSF-G250 chimericmolecule. The molecule can be provided in solution, in suspension, or asa (e.g. lyophilized) powder. The GM-CSF-G250 may be packaged withappropriate pharmaceutically acceptable excipient and/or adjuvant, e.g.in a unit dosage form.

[0173] Similarly, kits optimized for DNA vaccination of a constructencoding a GM-CSF-G250 polypeptide preferably comprise a containercontaining a GM-CSF-G250 nucleic acid (e.g. a DNA). As with thepolypeptide, the nucleic acid can be provided in solution, insuspension, or as a (e.g. lyophilized) powder. The GM-CSF-G250 nucleicmay be packaged with appropriate pharmaceutically acceptable excipientand/or facilitating agent(s), e.g. in a unit dosage form. The kit canfurther include reagents and/or devices to facilitate delivery of thenucleic acid to the subject (e.g. human or non-human mammal).

[0174] Kits optimized for adoptive immunotherapy typically include acontainer containing a chimeric GM-CSF-G250 polypeptide as describedabove. The kits may optionally include a nucleic acid (e.g. a vector)encoding a GM-CSF-G250 fusion protein for ex vivo transfection of cells.Such kits may also, optionally, include various cell lines (e.g. RCC)and/or reagents (e.g. IL-2) to facilitate expansion of activated cells.

[0175] The kits can, optionally, include additional reagents (e.g.buffers, drugs, cytokines, cells/cell lines, cell culture media, etc.)and/or devices (e.g. syringes, biolistic devices, etc.) for the practiceof the methods of this invention.

[0176] In addition, the kits may include instructional materialscontaining directions (i.e., protocols) for the practice of the methodsof this invention. Thus typical instructional materials will teach theuse of GM-CSF-G250 chimeric molecules (or the nucleic acid encodingsuch) as vaccines, DNA vaccines, or adoptive immunotherapeutic agents inthe treatment of renal cell cancers. While the instructional materialstypically comprise written or printed materials they are not limited tosuch. Any medium capable of storing such instructions and communicatingthem to an end user is contemplated by this invention. Such mediainclude, but are not limited to electronic storage media (e.g., magneticdiscs, tapes, cartridges, chips), optical media (e.g., CD ROM), and thelike. Such media may include addresses to internet sites that providesuch instructional materials.

EXAMPLES

[0177] The following examples are offered to illustrate, but not tolimit the claimed invention.

Example 1 Cloning and Expression of GM-CSF and G250 Fusion Protein.

[0178] This example describes the cloning, expression and purificationof a GM-CSF-G250 fusion protein.

[0179] Cloning of Human GM-CSF-G250 Fusion Gene

[0180] A full-length human GM-CSF cDNA (0.8 kb) was cleaved from plasmidp91023(B) vector (Wong et al. (1985) Science 228: 810-815) with Eco RIrestriction endonuclease. Eco RI (5′) and Not I sites (3′) were insertedinto the GM-CSF cDNA by PCR (0.4 kb) using a first primer:gcgggaattc(atg)tggctgcagagc (5′ GM-CSF Eco RI underlined, SEQ ID NO:5)and a second primer: gagggaggcggccgc(ctc)ctggactggctc (3′ GM-CSF Not Iunderlined, to remove stop codon, SEQ ID NO:6).

[0181] NotI (5′) and His-Stop-Gb1 II (3′) sites were introduced intofull length G250 cDNA (1.6 kb) by PCR using the following primers: 5′G250 (NotI): gagggagcggcc(gct)cccctgtgcccc (remove start codon, SEQ IDNO:7), 3′ G250-His-stop codon-Bgl II: (1)gcagaggtagagatct(cta)atggtgatggtgatggtgggctccagtctcggctacctc (SEQ IDNO:8; brackets=stop last, second underline=8 His), (2)ggagagatct(cta)atgatgatgatgatgatgatgatgggctccagtctcggctacctct (SEQ IDNO:9, brackets—stop last, second underline=8 His).

[0182] Fragments were ligated producing M-CSF-NotI-G250-His-stop-Bgl IIas follows:

[0183] 5′ (gag)ggcggcc(gct)cccctgtgcccc (rest of Gm-CSF-G250) (SEQ IDNO:10; where (gag) is the last of GM-CSF, and (gct) is the first ofG250.

[0184] The 250 fusion gene was inserted into the polyhedrin genelocus-base baculovirus transfer vector pVL 1393 (PharMingen). Inparticular, the plasmid pVL 1393 was cut with Eco RI and Bgl IIrestriction endonucleases and the Eco RI-GM-CSF-G250-his-stop-Bgl IIconstruct was inserted into the cut vector.

[0185] Insect cells (sf8 cells) were transfected using the BaculoGoldtransfection kit (Pharmingen). This involved co-transfection oflinearized BaculoGold virus DNA and recombinant plasmid DNA containingGM-CSF-G250 fusion gene into insect cells (sf8 cells). The recombinantbaculoviruses were amplified and the plaques were assayed to titer thevirus.

[0186] The G250-GM-CSF protein was purified according to protocolsprovided in the PharMigen Instruction Manual, 4th Edition, July 1997,page 41. Briefly beads were prepared by resuspending the Ni-NTA agarosebeads. Two ml of beads were poured into 10 ml chromatography column(binding approximately 7.5-15 mg of 6× His fusion protein). The beadswere allowed to settle in the column and the ethanol preservative wasdrained. The beads were then washed with 6× His wash buffer (Cat #21472A, PharMigen) with 7.5 ml of 6× His wash buffer twice.

[0187] A cell lysate preparation was prepared by resuspending a cellpellet in ice-cold insect cell lysis buffer (Cat # 21425A, PharMigen)containing reconstituted protease inhibitor cocktail (Cat# 21426Z,PharMigen). The cells were lysed on ice for 45 minutes using 1 ml oflysis buffer per 2×10⁷ cells). The lysate was transferred into a cleancentrifuge tube and centrifuged at 10,000 rpm for 30 minutes or filteredthrough a 0.22 μm filter. The supernatant was saved for the column andthe pellet was discarded. 150 μl lysate was saved for an SDS-PAGE gel orWestern blot and protein concentration determination.

[0188] Lysate was added to equilibrated Ni-NTA agarose beads foraffinity purification on a column. The supernatant was loaded slowly orthe beads were bound in a 15 ml conical tube with the lysate for 1 hourat 4° C. The flow-through fraction was saved.

[0189] The column was washed with 10-15 ml of 6× His wash buffer (Cat #21472A, PharMigen) and the column was allowed to drain without drying.The wash step was repeated until the wash A₂₈₀ was less than 0.01(approximately 4 washes).

[0190] The fusion protein was then eluted with imidazole. Briefly 4.5 mlof the 6× His elution buffer (Cat # 21476A, PharMigen) includingimidazole was added as follows:

[0191] i. 0.1 M imidazole with 6× His elution buffer;

[0192] ii 0.2 M imidazole with 6× His elution buffer;

[0193] iii 0.3 M imidazole with 6× His elution buffer;

[0194] iv 0.4 M imidazole with 6× His elution buffer;

[0195] v 0.5 M imidazole with 6× His elution buffer;

[0196] The elution speed was maintained at a rate less than or equal to1 ml per minute. The eluted fractions were collected (200 μL).

[0197] After analysis with SDS-PAGE gel and Western Blot, the clean andcorrect fractions were picked and pooled, dialyzed against PBS and theNi-NTA purification repeated again.

[0198] Once more after analysis with SDS-PAGE gel and Western Blot, theclean and correct fractions were picked and pooled, dialyzed againstPBS. Further purification was performed on a Q-sepharose column. 1.0 mlof the column was equilibrated with 50 mM NaCl Buffer X (20 mM Hepes, 1mM EDTA, 20% Glycerol, and 0.5 mM PMSF). The protein sample was loadedonto the column and the flow-through fraction was collected.

[0199] Further purification was performed on a SP-sepharose column. 1.0ml of SP-sepharose column was loaded with 50 mM NaCl BufferX (20 mMHepes, 1 mM EDTA, 20% Glycerol, and 0.5 mM PMSF). The protein sample wasloaded onto the column and the flow-through fraction was collected andsaved. The column was eluted with the gradient salt buffer (50 mM NaClBuffer X-1000 mM fraction are 5.0 ml and 0.1 ml, respectively). Elutionwas with 1000 mM NaCl buffer X.

[0200] The correct fractions were pooled and dialyzed against PBS.

[0201]FIG. 1 illustrates a RT-PCR analysis of RCC tumor cells. FIG. 2illustrates FACS analysis of dendritic cells derived from adherent PBMCcultures. FIG. 3 illustrates upregulation of HLA antigen in dendriticcells by GM-CSF-G250 fusion protein. FIG. 4 illustrates cytotoxicity ofbulk PMBC modulated by G250-GM-CSF fusion protein (patient 1). TABLE 1shows phenotypic modulation of bulk peripheral blood monocytes byGM-CSF-G250 fusion protein, IL-4, and IL-2 (patient #1). Phenotype Day 7Day 21 CD3⁺CD56⁻ 60 94 CD3⁻CD56⁺ 10 3 CD3⁺CD8⁺ 21 25 CD3⁺CD4⁺ 40 68CD3⁺TcR⁺ 54 90 CD3⁺CD25⁺ 10 25

[0202] TABLE 2 shows phenotypic modulation of bulk peripheral bloodmonocytes by GM-CSF-G250 fusion protein, IL-4, and IL-2 for patient #2.Phenotype Day 7 Day 21 Day 42 CD3⁺CD56⁻ 70 90 100 CD3⁻CD56⁺ 13 1 0CD3⁺CD8⁺ 21 22 17 CD3⁺CD4⁺ 48 74 86 CD3⁺TcR⁺ 62 91 97 CD3⁺CD25⁺ 10 28 16

Example 2 Induction of G250 Targeted and T-Cell Mediated Anti-tumorActivity Against Renal Cell Carcinoma Using a Chimeric Fusion ProteinConsisting of G250 and Granulocyte-Monocyte Colony Stimulating Factor

[0203] Immunotherapy targeting the induction of a T-cell mediatedanti-tumor response in patients with renal cell carcinoma (RCC) holdssignificant promise. Here we describe a new RCC vaccine strategy thatallows for the concomitant delivery of dual immune activators: G250, awidely expressed RCC associated antigen, and granulocyte-macrophagecolony-stimulating factor (GM-CSF), an immunomodulatory factor forantigen presenting cells (APC). The G250-GM-CSF fusion gene wasconstructed and expressed in SF-9 cells using a baculovirus expressionvector system. The 66 kDa fusion protein (FP) was subsequently purifiedthrough a 6× His-Ni-NTA affinity column and a SP Sepharose/FPLC. Thepurified FP possessed GM-CSF bioactivity, comparable to that ofrecombinant GM-CSF when tested in a GM-CSF dependent cell line. Whencombined with IL-4 (1000 U/ml), FP (0.34 mg/ml) induced differentiationof monocytes (CD14⁺) into dendritic cells (DC) that express surfacemarker characteristic for APC. Up-regulation of mature DC (CD83+CD19−)(17% vs 6%) with enhanced HLA class I and class II antigen expressionwas detected in FP cultured DC as compared to DC cultured withrecombinant GM-CSF. Treatment of PBMC with FP alone (2.7 mg/107 cells)augmented both Th₁ and Th₂ cytokine mRNA expression (IL-2, IL-4, GM-CSF,IFN-γ and TNF-α). When compared to various immune manipulationstrategies in the long-term cultures of bulk PBMC, cells treated with FP(0.34 mg/ml) plus IL-4 (1000 U/ml) for one week and then re-stimulatedwith FP weekly plus IL-2 (20IU/ml) induced the most growth expansion ofT cells expressing T cell receptor (TcR). Moreover, under suchimmunomodulatory manipulation, RCC specific cytotoxicity that could beblocked by anti HLA class I, anti-CD3 and anti-CD8 antibodies wasdemonstrated in four out of six tested PBMC cultures. In one testedpatient, an augmented cytotoxicity against lymph node (LN) derived RCCtarget was determined as compared to that against primary tumor targets,which corresponded to an eight-fold higher G250 expression in LN tumoras compared to primary tumor. The replacement of FP with recombinantGM-CSF completely abrogated the selection of RCC specific killer cells.All FP modulated PBMC cultures with antitumor activity showed anup-regulated CD3⁺CD4⁺ cell population. These results indicate thatGM-CSF-G250 FP is a potent immunostimulant with the capacity foractivating immunomodulatory DC and inducing a T-helper cell supported,G250 targeted, and CD8⁺ mediated anti-tumor response. These findingshave important implications for the use of GM-CSF-G250 FP as a tumorvaccine for the treatment of patients with advanced kidney cancer.

[0204] Introduction

[0205] Metastatic renal cell carcinoma (mRCC) poses a therapeuticchallenge because of its resistance to conventional modes of therapysuch as chemotherapy and radiation therapy (Figlin (1999) J. Urol. 61:381-387). Advances in the treatment of mRCC have evolved significantlyin the last decade since the FDA approval of interleukin-2 (IL-2) in1992. It has become clear that immunotherapy is capable of producingdurable remissions in selected RCC patients, yet the overall responserates of immunotherapy remain approximately 25% at best (Fisher et al.(1997) Cancer J. Sci. Amer., 3: S70) at the cost of measurabletoxicities to the patient. The recent identification of MHC restrictedtumor-associated antigens (TAA) and the understanding of the criticalrole of immunomodulatory dendritic cells (DC) have provided therationale for the development of tumor vaccines for cancer therapy (Wanget al. (1999) J. Mol. Med., 77:640-655; Xu et al. (2000) Trends inBiotech. 18: 167-172). Many cancer vaccine strategies have been designedand tested in both animal models and human trials with encouragingresults. These include peptide-based vaccines (Rosenberg et al. (1999)J. Immunol., 163:1690-1695; Parkhurst et al. (1996) J. Immunol., 157:2539-2548), dendritic cell (DC)-based vaccines (Yang et al. (2000) J.Immunol., 164: 4204-4211; Condon et al. (1996) Nature Med., 2:1122-1128;Zhou et al. (1996) Human Gene Ther., 10: 2719-2724; Nestle et al. (1998)Nature Med., 4: 328-332; Mulders et al. (1999) Clin. Cancer Res.,5:445-454), recombinant viruses/DNA/RNA based vaccines (Uhner et al.(1998) J. Virol., 72: 5648-5653; Ying et al. (1999) Nature Med., 5:823-827; Liu (1998) Nature Med., 4: 515-519), and gene modified tumorcells (Mach et al. (1999) Cancer Res., 60: 3239-3246). Despite that thefact that RCC is thought to be a relatively immunogenic tumor, no RCCassociated antigens have been identified and characterized inassociation with a significant rationale for the development of a kidneycancer targeted tumor vaccine (Gaugler et al. (1996) Immunogenetics,44:323-330; Brändle et al. (1996) J. Exp. Med., 183:2501-2508; Brossartet al. (1998) Cancer Res., 58: 732-736).

[0206] The first widely expressed RCC tumor associated antigen thatcontains HLA-A2 restricted CTL epitopes has been recently identified andcloned from a RCC cell line (Grabmaier et al. (2000) Intl. J. Cancer,85: 865-870; Vissers et al. (1999) Cancer Res., 59:5554-5559). This RCCassociated transmembrane protein, designated as G250, has been proven tobe identical to MN/CAIX, a TAA expressed in cervical cancer (Opavsky etal (1996) Genomics, 33: 480-487). Immunohistochemical staining withmAbG250 revealed that more than 75% of primary and metastatic RCCexpressed G250 while little to no expression was detected in the normalkidney (Grabmaier et al. (2000) Intl. J. Cancer, 85: 865-870). Inaddition, G250 expression is found in nearly all clear cell cancers ofthe kidney, the most common RCC variant, which provides further basisfor the use of G250 as a significant immune target for anti-cancertherapy. Antigen presentation is a crucial first step for vaccine-basedimmunotherapy. We therefore hypothesized that a chimeric proteinconsisting of G250 and GM-CSF, an immunomodulatory factor for thegeneration of functional DC, would augment vaccine capacity as comparedto the use of either agent alone. Several chimeric fusion proteinscontaining GM-CSF have been reported and have shown a variety of complexbiological effects dependent on their fusion components (Hall et al.(1999) Leukemia, 13: 629-633; Tripathi et al. (1999) Hybridoma 18:193-202; Battaglia et al. (2000) Exp. Hematol., 28: 490-498; Batova etal. (1999) Clin. Cancer Res., 5: 4259-4263). GM-CSF has been wellcharacterized as a growth factor that induces the proliferation andmaturation of myeloid progenitor cells (Hill et al. (1995) J. LeukocyteBiol. 58: 634-642). It enhances macrophage and granulocyte naturalcytotoxicity against tumor cells (Parhar et al. (1992) Europ. CytokineNetwork, 3: 299-306). The function of GM-CSF as a key factor for thedifferentiation of DC further substantiates its adjutant impact inimmune based vaccine therapy (Jonuleit et al. (1996) Archives ofDermatological Res. 289:1-8). Direct evidence of the adjuvant effects ofGM-CSF in vaccine based immunotherapy has been demonstrated in animalmodels. Immunization with tumor peptide at skin sites containingepidermal DC newly recruited by pre-treatment with DNA encoding GM-CSFelicited an antigen specific T cell response, whereas peptideimmunization of control skin site showed no immune response (Bowne etal. (1999) Cytokines Cellu. Mol Ther., 5: 217-225). Likewise, treatmentof established tumor with a hybridized cellular vaccine generated byfusing GM-CSF gene-modified DC with melanoma cells showed a greatertherapeutic efficacy when compared to the treatment with hybridizedvaccine generated with non-modified DC (Cao et al. (1999) Immunol., 97:616-625). An initial Phase I trial further demonstrated that systemicinjection of GM-CSF and IL-4 was capable of inducing tumor regressionand stable disease response in patients with advanced RCC and prostatecancer (Roth et al. (2000) Cancer Res., 60:1934-1941). Similarly,vaccination of patients with irradiated autologous RCC or melanoma cellsengineered to secrete human GM-CSF also induced a potent anti-tumorimmunity (32 Simons et al. (1997) Cancer Res. 57: 1537-1546; Soiffer etal. (1998) Proc. Natl. Acad. Sci., USA, 5: 13141-13146).

[0207] In this example, we describe a strategy to generate fusionproteins (FP) consisting of G250 and GM-CSF. In addition, we tested thefeasibility of using this non-viral and non-cellular RCC tumor vaccineas an immunostimulant for the in vitro modulation of DC and induction ofG250 targeted anti tumor response in PBMC cultures, that were derivedfrom patients with advanced kidney cancer.

[0208] Materials and Methods

[0209] Cloning of GM-CSF-G250 Fusion Gene in pVL 1393 Vector

[0210] Plasmid p91023(B)-GM-CSF (Wong et al. (1985) Science 228:810-815) was digested with EcoR I, and the 0.8 kb fragment containingthe full length of GM-CSF cDNA was used to generate the 0.4 kb GM-CSFfragment containing the functional epitope flanked by an EcoR I site onthe 5′ side and a Not I site on the 3′ side replacing the GM-CSF stopcodon, by DNA PCR. The GM-CSF fragment from the PCR product wassubcloned into the EcoR I and Bgl II sites of polyhedrin genelocus-based baculovirus transfer vector pVL1393 (Pharmigen, San Diego,Calif.). Similarly, pBM20CMVG250 Osterwick (2000) Int. J. Cancer, 85:A65-A70) was used to amplify the full length of G250 cDNA (1.6 kb)containing a Not I site followed by a 6 nucleotide linker coding for 2arginines by PCR in the 5′-flanking region of the G250 after the removalof its start codon. The 3′-flanking region of G250 was designed toencode 6 histidines followed by the stop codon and Bgl II site. The G250fragment was gel purified. Both the vector pVL1393 already contained theGM-CSF and the G250 PCR amplified fragments were cut out with Not I andBgl II. The G250 fragment and the vector were ligated for 3 hr at 16°C., and later transformed and plated on LB plates. The coloniescontaining the correct plasmid were purified by cesium chloride buoyantultracentrifugation. The plasmid was cut with a set of differentrestriction enzymes to verify the plasmids. The plasmid clones werefurther verified for histidine tag using an Amplicycle Sequencing Kit(Perkin Elmer).

[0211] Generation and Purification of Fusion Protein

[0212] The recombinant baculovirus containing His-tagged GM-CSF-G250fusion gene was generated by co-transfection of 0.5 mg BaculoGold DNA(modified AcNPV baculovirus DNA) (Pharmigen, San Diego, Calif.) and 5 mgof pVL1393/GM-CSF-G250 in Sf9 cells (Spodoptera frugiperda). Viruseswere further amplified at a low MOI (<1) in adherent Sf9 insect cellsand the titers of the virus were determined by plaque assay. Expressionof GM-CSF-G250 FP in Sf9 cells was determined by immunocytochemicalanalysis using anti-G250 mAb, anti-GM-CSF antibody (Genezyme, Cambridge,Mass.) and irrelevant Ab. Sf9cells infected with pVL1392-XylErecombinant virus (Pharmigen) and uninfected Sf9cells were used asnegative control for FP expression and analysis. The viruses used forprotein production were isolated and amplified from a single plaque.Cell lysate was prepared from the Sf9 cells infected with viruses at MOIof 5 for three days with insect cell lysis buffer containing proteaseinhibitor cocktail (Pharmigen, San Diego, Calif.). Filtered lysate (0.22mm filter) was applied to a Ni²+-NTA agarose column with high affinityfor 6× His (Qiagen, Santa Clarita). After extensive washing of thecolumn (50 mM Na-phosphate, 300 mM NaCl, 10% glycerol, pH 8.0), thefusion protein was eluted stepwise column (50 mM Na-phosphate, 300 mMNaCl, 10% glycerol, pH 6.0) by increasing concentration of imidazolefrom 0.1M up to 0.5M. All purification steps were carried out at 4° C.Fractions were analyzed by Western blot using anti-GM-CSF antibody. Thepeak fractions were combined, dialyzed and re-applied to an Ni²⁺-NTAagarose column for repeated purification. Fractions containing FP werepooled, dialyzed and further applied to a FPLC column containing SPSepharose, High Performance (Amersham Pharmacla Biotech, Piscataway,N.J.). Fusion protein was eluted with an increasing salt gradient from50 mM to 1M NaCl in buffer X (20 mM Tris, 1 mM EDTA, 10% Glycerol) andthe fractions containing FP were pooled, dialyzed and sterilized through0.2 m filter. The Coomassie blue and silver stains were used to analyzethe purity of GM-CSF-G250 fusion protein. The protein concentration wasdetermined by Bio-Rad Dc Protein Assay (Bio-Rad, Hercules, Calif.94547).

[0213] GM-CSF Dependent Proliferation Assay

[0214] The biological activity of the GM-SF component of the FP wasdetermined by measuring the proliferation of GM-SF dependent TF-cells(Kitamura et al. (1989) J. Cellu. Physiol., 140: 323-334) in thepresence of FP. TF-1 cells were seeded in 96-well plates in triplicatein culture medium (RPMI medium+10% FBS) at the concentration of 2×10⁴cells/well containing titrated concentration of FP or the correspondingamount of recombinant human GM-CSF (rh-GM-CSF). Cultures were incubatedfor 5 days and ³H-thymidine (0.1 mCi/well) was added 12 h prior toharvest. The incorporated ³H-thymidine was measured by scintillationcounting with a P counter.

[0215] Phenotypic Analysis of DC by Fluorescence Activated Cell Sorting(FACS)

[0216] The phenotype of DC generated from both adherent and bulk PBMCwas determined by two-color immunofluorescence staining as described inHinkel et al. (2000) J. Immunother., 23: 83-93. Both adherent PBMC andnon-fractionated bulk PBMC were cultured with 1000 U/ml of IL-4 pluseither GM-CSF (800 U/ml) or FP (0.34 mg/ml) for 7 days and the identityof DC was determined. Cell cultures (1×10⁵ cells) were re-suspended in50 ml FACS buffer (PBS, 2% new born calf serum, 0.1% sodium azide) andincubated with 10 ml of the appropriate fluorescein isothiocyanate(FITC) or phycoerythrine (PE) labeled monoclonal antibodies for 30 minat 4° C. After staining, cells were washed twice with PBS andre-suspended in 200 ml FACS buffer plus 200 ml paraformaldehyde 2%. Fiveto ten thousand events per sample were acquired on a Becton-DickinsonFACScan II flow cytometer that simultaneously acquires forward (FSC) andside scatter (SSC), as well as FL1 (FITC) and FL2 (PE) data, andanalyzed utilizing the CellQuest Software (Becton-Dickinson, San Jose,Calif.).

[0217] Settings for all parameters were optimized at the initiation ofthe study and were maintained constant throughout all subsequentanalyses. DC population in bulk PBMC culture was gated based on theirsize and granularity. In all samples the position of quadrant cursorswas determined by setting them on samples stained with the appropriatedisotype control antibody. The following antibodies were employed forcharacterization of the DCs phenotype: Anti-CD86 (B7-2; PharMingen,San-Diego, Calif.), Anti-CD40 (Caltag, Burlingame, Calif.), anti-HLAclass I (W6/32, ATCC HB95), anti-HLA-DR (Immunocytometry System; BectonDickinson, Mountain View, Calif.), anti-CD14 (Catlag laboratories, SanFrancisco, Calif.) and isotype control IgG1/IgG2a (Beckton Dickinson).The CD83⁺ surface marker was used to delineate the maturation of DC. Inorder to discriminate DC (CD83⁺CD19⁻) from activated B cells(CD83⁺CD19⁺), dual color staining utilizing CD19FITC and CD83PE(Immunotech, Marseille, France), was performed.

[0218] Semi-quantitative Reverse Transcriptase-polymerase Chain Reaction(RT-PCR) Analysis of Cytokine Profile in PBMC.

[0219] Total RNA was extracted from PBMC treated with FP (2.7 mg/107cells) for various time intervals up to 24 hr at 37° C., using acidguanidine isothiocyanate-phenol-chloroform extraction. Reversetranscription of messenger RNA into cDNA was carried out by incubatingtitrated RNA with AMV reverse transcriptase, primer oligo (dT), dNTP,and RNAse inhibitor at 42° C. for 1 hour. One ml of each cDNA sample wasamplified utilizing PCR in a total volume of 25 ml, (30 ng[³²P]-5′-oligonucleotide, 10 ng 3′-oligonucleotide primer, 2.5 mlmodified 10× PCR buffer, 1.25 units Taq polymerase, and autoclaveddouble distilled water to a volume of 25 ml). The PCR mixture wasamplified for 25 cycles in a DNA Thermocycler (Perkin-Elmer, Norwalk,Conn.). Each cycle consisted of denaturation at 94° C. for one minuteand annealing/extension at 65° C. for 2 minutes. The 32P-labeled PCRproducts were then visualized directly via acrylamide gelelectrophoresis and autoradiography and then quantitated by excision ofbands and subsequent scintillation counting.

[0220] The signal intensity of each amplified product was calibrated toits corresponding β-actin mRNA expression as an internal control forquantitation of expression levels. In addition, quantitative analysiswas further elucidated by a serial dilution of mRNA (1:3, 1:10, 1:30 and1:300) and co-amplification of β-actin and GM-CSF mRNA. The sequences ofthe oligonucleotide primer pairs are as follows: β-actin: 5′-CAA CTC CATCAT GAA GTG TGA C-3′ (SEQ ID NO:11), 3′-CCA CAC GGA GTA CTT GCG CTC-5′(SEQ ID NO:12); GM-CSF: 5′-CCA TGA TGG CCA GCC ACT AC-3′ (SEQ ID NO:13),3′-CTT GTT TCA TGA GAG AGC AGC-5′ (SEQ ID NO:14), TNF-α: 5′-TCT CGA ACCCCG AGT GAC AA-3′ (SEQ ID NO:15), 3′-TAC GAC GGC AAG GAT TAC ATC-5′ (SEQID NO: 16); IFN-γ: 5′-ATG AAA TAT ACA AGT TAT ATC TTG GCT TT-3′ (SEQ IDNO:17), 3′-ATG CTC TTC GAC CTC GAA ACA GCA T-5′ (SEQ ID NO:18); IL-2:5′-GGA ATT AAT AAT TAC AAG AAT CCC-3′ (SEQ ID NO:19), 3′-GTT TCA GAT CCCCTT TAG TTC CAG-5′ (SEQ ID NO:20); IL-4: 5′-CTT CCC CCT CTG TTC TTC CT,3′ TTC CTG TCG AGC CGT TTC AG-3′ (SEQ ID NO:21).

[0221] Immunomodulation of PBMC with Fusion Protein

[0222] Fresh isolated PBMC from patients with RCC expressing G250 werecultured in RPMI 1640 medium supplemented with 10% autologous serum.Various schedules of immunomodulatory protocols of PBMC cultures with FPwere carried out as described in Table 3 and FIG. 10. The growth of PBMCwas determined by cell count, and the cytolytic activity of PBMCs wasassayed for different targets in a prolonged 18-hour chromium-51 (51 Cr)release assay. Five thousand 51 Cr-labeled target cells per well wereseeded in a 96-well microtiter plate (Costar, Cambridge, Mass.) andmixed with PBMC yield several E/T ratios (40:1, 20:1, 10:1, and 5:1).Cytotoxicity was expressed as lytic units (LU) per 10⁶ effector cellswith lytic unit being defined as the number of effector cells thatinduce 30% lysis. T cell mediated and RCC specific cytotoxicity wasconfirmed by blocking assays in which targeted autologous tumor cellswere pretreated with anti-human leukocyte antigen (HLA) class I, classII, or PBMC were pretreated anti-CD3, anti-CD4, anti-CD8, or isotypecontrol antibody (Becton Dickinson) for 30 min at 4° C., before additionof cells to cytotoxicity culture plates. Spontaneous release of alltargets was equal or less than 20% of maximal release of 51-Cr release.The following target cells were used: autologous normal kidney cells(G250−), autologous RCC tumor cells (G250+), allogeneic RCC cells(G250+), allogeneic prostate cells (CL-1), and human fibroblast (hFb).TABLE 3 Phenotypic Modulation of Bulk PBMC by Fusion Protein (FP) IL-2 +IL-4 + FP FP FP + IL-4 Phenotype Pre-cultured IL-2 IL-2 + FP IL-2 + FPIL-2 + FP IL-2 + FP CD56⁺CD3⁻ 25 13 11 4 9 1 CD56⁻CD3⁺ 46 47 70 84 88 94CD4⁺CD8⁻ 31 28 39 42 66 46 CD4⁺CD8⁺  3 10 20 29 4 28 CD4⁻CD8⁺ 22 24 3124 22 25 CD3⁺TcR⁺ 40 45 72 69 79 96 CD3⁺CD25⁺ 19 43 61 54 17 86

[0223] Results

[0224] Generation of GM-CSF-G250 Fusion Protein fromBaculovirus-infected SF-9 Cells.

[0225] Baculovirus expression technology and the 6× His affinitypurification system were used to generation GM-CSF-G250 FP as describedabove. The success of gene cloning and generation of recombinantbaculovirus was verified by the immunohistochemical staining of virusesinfected Sf9 cells using anti GM-CSF and anti-G250. Abundant G250 andGM-CSF protein expression were detected in Sf-9 cells that were infectedwith GM-CSF-G250 recombinant baculoviruses (FIG. 6A, top and middlepanel), whereas no expression of GM-CSF or G250 was detected innon-infected cells (FIG. 6A, bottom panel) or cells infected withpVL1392-XylE recombinant viruses (data not shown). Western blot analysiswas used to evaluate the efficiency of 6× His affinity tag in FP forNi2+-NTA agarose. An expected 66-kDa band which detected withanti-GM-CSF appeared in the fractions collected from number 5 to number25 with the peak concentration at fraction 15 to 19 (FIG. 6B). Theprotein purity was further improved by re-run of positive fractionsthrough Ni²⁺-NTA agarose column and subjected to FPLC using SP Sepharosecolumn. A major single 66 kDa band was detected in SDS-PAGE analysisstained with coomassie blue (FIG. 6C).

[0226] Purified GM-CSF-G250 Fusion Protein Retained GM-CSF Bioactivity

[0227] To determine whether the bioactivity of the GM-CSF was preservedin the purified FP, the FP was analyzed for its ability to support theproliferation of a GM-CSF dependent cell line, TF-1. Serial dilutions ofFP were performed to span the effective concentration range. Theexperiments were conducted in parallel with recombinant GM-CSF. Theresults from the ³H-thymidine incorporation assay demonstrated that theFP could stimulate TF-1 cell growth with a biphasic dose dependentmanner (FIG. 7B). When compared to recombinant GM-CSF (FIG. 7A),comparable bioactivity was determined in the presence of FP withequivalent concentrations of GM-CSF in the range between 0-6.71 ng/ml(=0-30.2 ng/ml FP). In the presence of concentrations higher then 30.2ng/ml of FP, the growth induction of TF-1 by FP exceeded the growthinduction by recombinant GM-CSF by 1.3 fold (FIGS. 7A, 7B).

[0228] Immunomodulatory Effect of Fusion Protein on Antigen PresentingCells in PBMC Culture

[0229] In order to study how the FP could affect the development of DC,PBMC derived from patients with RCC were cultured in the presence of FP(0.34mg/ml) plus IL-4 (1000 U/ml) for 7 days and compared to thatcultured in GM-CSF (800U/ml) plus IL-4. FACS analysis revealed a highpercentage of large granulocytes expressing B7-2⁺, CD40⁺ and HLA-DR⁺ inboth conditions, whereas CD14⁺ cells were negligible (FIG. 8A). However,when compared to dendritic cells cultured with recombinant cytokines, anenhanced expression of both HLA class I (mean relative linearfluorescence intensity=4830 vs 3215) and HLA class II (6890 vs 6290) wasdetected in the FP modulated DC cultures (FIG. 8B). In addition, therewas a three-fold increase of mature DC (CD83+CD19−) in FP modulated DCcultures (FIG. 8C). This observation was consistent in several bulk PBMCcultures derived from RCC patients (n=3) and healthy donors (n=2).Similar FP mediated immunomodulatory profile was also determined onconventional adherent DC cultures (data not shown). A lower efficiencyof DC differentiation was observed when DC were cultured in the presenceof FP alone without IL-4. A mix of CD14⁺ and CD14-B7-2⁺ cell populationwere determined on day 7 (data not shown).

[0230] Fusion Protein Induces Activation of Cytokine Genes in PBMC

[0231] To identify whether the fusion protein has a direct effect on theregulation of cytokine genes in PBMC, freshly isolated PBMC cells,derived from RCC patients, were treated with FP alone (2.7mg/10⁷ cells).The kinetics of cytokine gene activation was followed by analysis ofmultiple cytokine mRNA expression through time course as indicated inFIG. 9. When compared to untreated PBMC, treatment of uncultured PBMCwith FP gradually enhanced GM-CSF, TNF-α, IFN-γ, IL-4, IL-2 mRNAexpression with the peak level at 24 hr post treatment except IL-4. Thepeak of IL-4 mRNA expression was detected at 6 hr after the treatment(FIG. 9).

[0232] Fusion Protein Induces T Cell Mediated and G250 Targeted ImmuneResponse in PBMC Cultures

[0233] Five immunomodulatory protocols with and without FP were testedand compared in PBMC cultures. These culture conditions included 1) IL-2alone (40 IU/ml), 2) IL-2+FP (0.34 mg/ml)(re-stimulated weekly), 3)IL-2+IL-4 (1000 U/ml)+FP for one week then restimulated with FP+IL-2, 4)FP alone for one week then restimulated with FP+IL-2 and 5) FP+IL-4 forone week then re-stimulated with IL-2+FP. As indicated in FIG. 10(patient #1), among various immunomodulatory treatments tested, thecondition with pretreated PBMC with FP plus IL-4 for one week andsubsequently restimulated with IL-2 (40 IU/ml) and FP weekly, showed thehighest growth expansion (6.0×) (FIG. 10A). A similar growth profilewith enhanced growth activity in this particular condition wasdetermined in another 3 PBMC cultures that were derived from patientswith RCC. In one particular patient (patient#1) who had a positive lymphnode (LN), an enhanced cytotoxicity against LN derived tumor target wasdetermined in all four FP modulated PBMC cultures (3 cycles ofre-stimulation) when compared to the cytotoxicity against primary tumortarget (FIG. 10B). Notably, this enhanced killing activity correspondedto an eight-fold increase of G250 mRNA expression in LN derived RCCtumor, as determined by a semi-quantitative RT-PCR, when compared toprimary RCC cells (FIG. 10C). When LN tumor target cells were pretreatedwith anti HLA class 1 (77%) or alternatively, effectors were pretreatedwith anti CD3 (66%) or anti CD8 (55%) prior to the assay, RCC targetedcytotoxicity was markedly reduced. Whereas anti HLA class II (33%) oranti CD4 (33%) treatment could lead only to a lesser inhibition ofcytotoxicity (FIG. 10C). Although poor growth expansion (1.8×) wasdetected in the condition that pretreated PBMC with FP alone for oneweek and re-stimulated with IL-2 plus FP, the highest cytotoxcityagainst both primary and LN derived RCC target was detected whencompared to other tested conditions (FIG. 10B).

[0234] To identify the phenotypic identity of FP modulated PBMC thatpossess anti-tumor activity, phenotypic analysis was performed on theday when cytotoxicity was determined. A markedly increased T cellpopulation (70-94%) expressing T cell receptor (72-96%) was detected inall FP stimulated PBMC cultures, when compared to pre-cultured PBMC(46%) or PBMC cultured with IL-2 alone (47%) (Table 3). Notably, the Tcell population expressing the most IL-2 receptor (CD3⁺CD25⁺) (86%) wasdetermined to occur in the condition that pretreated cells with FP plusIL-4 prior to re-stimulation with IL-2 and FP. This also corresponded tothe greatest growth expansion in T cell population when compared to allother tested immunomodulatory protocols (FIG. 10A). Correspondingly,PBMC that were pretreated with FP for one week demonstrated a minimal Tcell population expressing the IL-2 receptor (19%) and demonstrating theleast growth expansion (Table 3 and FIG. 10A).

[0235] Replacement of FP with GM-CSF Abrogates the Selection of RCCTargeted Cytotoxic T Cells

[0236] In order to confirm that the component of G250 in the FP is thedeterminant for the growth selection of CTL against RCC, cytotoxicityassay was performed with PBMC that were cultured in the presence ofGM-CSF and IL-4 for one week then continuously restimulated with IL-2and GM-CSF (800 U/ml). Minimal cytotoxicity against autologous RCC wasdetermined in all tested PBMC cultures without FP stimulation. Whereasthe corresponding PBMC cultures stimulated with FP showed an MHCrestricted, T cell mediated cytotoxicity against autologous RCC (FIG.11A) that expressed high level of G250 (data not shown). PredominantCD3⁺CD4⁺ cell population was detected in all three FP modulated PBMCcultures (68%, 74%, and 66%) that expressed antitumor activity, whencompared to CD3⁺CD8⁺ cell population (25%, 22%, and 30%) (FIG. 11B).Moreover, both Th1 and Th2 cytokine mRNA were detected in these FPmodulated PBMC cultures which included GM-CSF, TNF-α, IFN-γ, IL-2 andIL-4 (data not shown).

[0237] Discussion

[0238] Renal cell carcinoma (RCC) is responsive to immunotherapy.However, it is believed that no immune-based treatment protocol has beenpreviously shown that would effectively eradicate tumor lesions in themajority of patients. It is believed that means of immune strategy, thetype of immune activators used, the method of administration, and thepretreatment immune status of patients all could influence the ultimateimmune response in cancer patients that are treated with immune-basedtherapy. Therefore, an important issue for an effective cancer vaccineis the development of a potent adjuvant that can facilitate bothinduction and augmentation of an immune response with antitumoractivity. To achieve this, we proposed a chimeric construct consistingof G250 and GM-CSF. The demonstration of G250 expression in SF-9 cellsand GM-CSF bioactivity in the purified 66 kDa band of protein moleculeconfirmed the efficacy of the gene construct and the effectiveness ofthe selected protein purification method.

[0239] Antigen presentation by DC is important, not only for theinduction of primary immune responses, but may also be important for theregulation of the type of T cell-mediated immune response (Banchereau etal. (2000) Ann. Rev. Immunol., 18: 767-811). We recently developed anon-fractionated bulk PBMC culture system for the study of thematuration and immunomodulatory function of CD14⁺ derived DC and theinteraction between the DC and co-cultured lymphocytes (Hinkel et al.(2000) J. Immunother., 23: 83-93). Using this system, antigen loadingcan be performed during the early culture period of PBMC in the presenceof GM-CSF and IL-4, when immature DC/monocytes can take up and processtumor antigen. As we have previously demonstrated that DC modulatedco-cultured lymphocytes in bulk PBMC culture can be further expanded toCTL by repetitive stimulation with low dose IL-2 and RCC tumor lysate.Likewise, direct treatment of bulk PBMC with IL-4 and FP not onlyinduced the differentiation of CD14⁺ cells into DC but also increasedthe maturation of DC when compared to DC generated in IL-4 and GM-CSF.This suggests that signaling pathway in DC maturation can be induced byFP stimulation (Rescigno et al. (1998) J. Exp. Med., 188: 2175-2180).Moreover, when compared to recombinant GM-CSF, up-regulated HLA antigenexpression was determined on FP modulated DC further indicating that DCare capable of internalizing, processing, and presenting FP through HLAantigens in DC. Whether the G250 was taken up by APC through GM-CSFreceptor internalization or via the G250 component remains to bedetermined.

[0240] It appears that preincubation of PBMC with FP and IL-4 prior toexposing IL-2 facilitates a better effector expansion. This may beexplained if exogenous IL-4 could synergize the FP for the mobilizationof DC differentiation and maturation and subsequent presentation of theantigen peptides to the surrounding immune cells. Although a successfulCTL selection also could be achieved by other tested immunomodulatoryprotocols with FP, the growth expansion of CTL was not favorable. Thismay be partly associated with a “delayed” DC differentiation under thesub-optimal concentration of IL-4 (note: FP can induce IL-4 secretion byPBMC). Moreover, pre-exposure of IL-2 to a non-antigen stimulated PBMCusually results in the expansion of non-specific lymphokine activatedkiller cells with short-term killing activity (Roussel et al. (1990)Clin. Exp. Immunol., 82: 416-421). Recently, Huang et al. (1994)Science, 264: 961-965, demonstrated that even “immunogenic” tumors, suchas those modified to express co-stimulatory molecules, fail to stimulatethe immune system, unless functional APC are available to process andpresent the antigens. It thus appears that the most effective anticancer vaccine strategy should target manipulation of enhancing T cellpriming at the level of APC in patients.

[0241] That the replacement of FP with equivalent dose of recombinantGM-CSF abrogated the selection and propagation of RCC specific CTLsuggests that activation and propagation of CTL is antigen(G250)-dependent. Whereas the GM-CSF has served as an effective adjuvantfor antigen presentation and amplification of T cell activity includingcytokine response (Mach et al. (1999) Cancer Res., 60: 3239-3246;Pulendran et al. (1999) Proc. Natl. Acad. Sci., USA, 96:1036-1041).Although FP induced G250 targeted antitumor activity is mainly mediatedby CD8⁺T cells, a predominant up-regulation of CD4⁺T cells was detectedin most cultures. The FP mediated Th1 and Th2 cytokine release andenhancement of HLA class II expression in DC cells further suggests FPmediated antitumor immunity may involve the priming of both CD4⁺ andCD8⁺T cells specific for G250. The role of CD4⁺T helper cells in thisresponse may be attributed to provide regulatory signals required forthat priming of MHC class I restricted CD8⁺CTL (Mach et al. (1999)Cancer Res., 60: 3239-3246).

[0242] Studies comparing the efficacy of various formulations of tumorvaccines in parallel demonstrated that the use of DC transfected withDNA coding for TAA is superior to peptide-pulsed DC and naked DNA basedvaccine for eliciting both antigen-specific CD8 and CD4T cell response(Yang et al. (1999) Intl. J. Cancer, 83: 532-540). This observationindicates that computer predicted peptides might not be naturallyprocessed and presented on the tumor cells surface for the recognitionby peptide reactive T cells. Thus, some in-vitro peptide-reactive Tcells could only lyse peptide pulsed cell targets but not tumor cellsexpressing the entire tumor antigen (Vissers et al. (1999) Cancer Res.,59:5554-5559; Rammensee et al. (1993) Annual Rev. Immunol., 11:213-244). Likewise, a peptide-based vaccine could effectively elicitexpansion of vaccine specific T cells in PBMC of cancer patients, butsuch response was not associated with a clinical tumor regression (Leeet al. (1999) J. Immunol., 163: 6292-6300). Therefore, immunization withthe current construct of whole G250 antigen may have the advantage overthe peptides for the presentation of multiple, or unidentified epitopesin association with MHC class I and class II molecules by APC. On thebasis of the potency and specificity of the GM-CSF-G250 fusion proteinin the activation of G250-reactive T cells with antitumor activity, ourdata indicate that vaccination with GM-CSF-G250 FP will providetherapeutic impact for the treatment of advanced kidney cancer.

Example 3 Generation of the Mammalian Expression Vector pCEP4-GMCSF-G250

[0243] i) Amplification of the Recombinant Gene GMCSF-G250 Without theHis Tag and Cloning into pGEM-T.

[0244] pVL1393-GMCSF-G250 (His tag) was used as a template in a PCRreaction that was carried out using primers designed to introduce a KpnIbefore the start codon of the GMCSF (5′ primer) and a XhoI site afterthe stop codon (3′ primer). In addition, the 3′ primer was designed toeliminate the poly-Histidine coding sequence previously introduced fordetection and purification purposes. A high fidelity amplificationsystem (Expand High Fidelity System, Boehringer-Mannheim) was used toavoid mutations in the PCR product, which was directly cloned intopGEM-T vector (Promega), a convenient vector for further sequencing andcloning steps, resulting in pGEMT-GMCSF-G250. Completely sequencing ofthe GMCSF-G250 gene revealed no mutation and the expected absence of thepoly-histidine coding sequence.

[0245] ii) Cloning of the GMCSF-G250 into the Mammalian ExpressionVector pCEP4.

[0246] pCEP4 is an episomal vector mammalian expression vector that usesthe cytomegalovirus (CMV) immediate early enhancer/promoter for highlevel transcription of recombinant genes inserted into the multiplecloning sites and also carries the hygromycin B resistance gene forstable selection in transfected cells. Subcloning of GMCSF-G250 intopCEP4 was carried out with a digestion of vectors pGEMT-GMCSF-G250 andpCEP4 with restriction enzymes KpnI and XhoI and further gelpurification and ligation of the resulting linearized pCEP4 andGMCSF-G250. The new plasmid pCEP4-GMCSF-G250 (FIG. 12) contained therecombinant gene in the proper orientation as expected. A SalI digestionof pCEP4-GMCSF-G250 released the complete expression cassette CMVpromoter-gene-polyadenylation signal (3.7 kb, FIG. 13) that can becloned into the E1 and E3 deleted adenovirus or gutless adenovirusbackbone for generation of fusion gene recombinant adenovirus. Thesefusion gene recombinant viruses can be used as a virus-form to immunizepatients directly or alternatively, to infected RCC cells or DC togenerate kidney cancer vaccine for the direct immunization of patients.Alternatively, defined RCC cell lines can be stably transfected withpCEP4-GMCSF-G250 and used as RCC tumor vaccine. These various types ofG250-GM-CSF vaccine formulations also can be used as an in-vitroimmunostimulant for activation and propagation of G250 targeted CTL fromPBMC or TIL cultures, which derived from patients with RCC then,re-infuses these CTL back to patients as an adoptive immunotherapy.

[0247] It is understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and scope of the appended claims. All publications, patents,and patent applications cited herein are hereby incorporated byreference in their entirety for all purposes.

1 22 1 610 PRT Artificial Sequence G250-GM-CSF fusion protein 1 Met TrpLeu Gln Ser Leu Leu Leu Leu Gly Thr Val Ala Cys Ser Ile 1 5 10 15 SerAla Pro Ala Arg Ser Pro Ser Pro Ser Thr Gln Pro Trp Glu His 20 25 30 ValAsn Ala Ile Gln Glu Ala Arg Arg Leu Leu Asn Leu Ser Arg Asp 35 40 45 ThrAla Ala Glu Met Asn Glu Thr Val Glu Val Ile Ser Glu Met Phe 50 55 60 AspLeu Gln Glu Pro Thr Cys Leu Gln Thr Arg Leu Glu Leu Tyr Lys 65 70 75 80Gln Gly Leu Arg Gly Ser Leu Thr Lys Leu Lys Gly Pro Leu Thr Met 85 90 95Met Ala Ser His Tyr Lys Gln His Cys Pro Pro Thr Pro Glu Thr Ser 100 105110 Cys Ala Thr Gln Ile Ile Thr Phe Glu Ser Phe Lys Glu Asn Leu Lys 115120 125 Asp Phe Leu Leu Val Ile Pro Phe Asp Cys Trp Glu Pro Val Gln Glu130 135 140 Ala Ala Ala Pro Leu Cys Pro Ser Pro Trp Leu Pro Leu Leu IlePro 145 150 155 160 Ala Pro Ala Pro Gly Leu Thr Val Gln Leu Leu Leu SerLeu Leu Leu 165 170 175 Leu Met Pro Val His Pro Gln Arg Leu Pro Arg MetGln Glu Asp Ser 180 185 190 Pro Leu Gly Gly Gly Ser Ser Gly Glu Asp AspPro Leu Gly Glu Glu 195 200 205 Asp Leu Pro Ser Glu Glu Asp Ser Pro ArgGlu Glu Asp Pro Pro Gly 210 215 220 Glu Glu Asp Leu Pro Gly Glu Glu AspLeu Pro Gly Glu Glu Asp Leu 225 230 235 240 Pro Glu Val Lys Pro Lys SerGlu Glu Glu Gly Ser Leu Lys Leu Glu 245 250 255 Asp Leu Pro Thr Val GluAla Pro Gly Asp Pro Gln Glu Pro Gln Asn 260 265 270 Asn Ala His Arg AspLys Glu Gly Asp Asp Gln Ser His Trp Arg Tyr 275 280 285 Gly Gly Asp ProPro Trp Pro Arg Val Ser Pro Ala Cys Ala Gly Arg 290 295 300 Phe Gln SerPro Val Asp Ile Arg Pro Gln Leu Ala Ala Phe Cys Pro 305 310 315 320 AlaLeu Arg Pro Leu Glu Leu Leu Gly Phe Gln Leu Pro Pro Leu Pro 325 330 335Glu Leu Arg Leu Arg Asn Asn Gly His Ser Val Gln Leu Thr Leu Pro 340 345350 Pro Gly Leu Glu Met Ala Leu Gly Pro Gly Arg Glu Tyr Arg Ala Leu 355360 365 Gln Leu His Leu His Trp Gly Ala Ala Gly Arg Pro Gly Ser Glu His370 375 380 Thr Val Glu Gly His Arg Phe Pro Ala Glu Ile His Val Val HisLeu 385 390 395 400 Ser Thr Ala Phe Ala Arg Val Asp Glu Ala Leu Gly ArgPro Gly Gly 405 410 415 Leu Ala Val Leu Ala Ala Phe Leu Glu Glu Gly ProGlu Glu Asn Ser 420 425 430 Ala Tyr Glu Gln Leu Leu Ser Arg Leu Glu GluIle Ala Glu Glu Gly 435 440 445 Ser Glu Thr Gln Val Pro Gly Leu Asp IleSer Ala Leu Leu Pro Ser 450 455 460 Asp Phe Ser Arg Tyr Phe Gln Tyr GluGly Ser Leu Thr Thr Pro Pro 465 470 475 480 Cys Ala Gln Gly Val Ile TrpThr Val Phe Asn Gln Thr Val Met Leu 485 490 495 Ser Ala Lys Gln Leu HisThr Leu Ser Asp Thr Leu Trp Gly Pro Gly 500 505 510 Asp Ser Arg Leu GlnLeu Asn Phe Arg Ala Thr Gln Pro Leu Asn Gly 515 520 525 Arg Val Ile GluAla Ser Phe Pro Ala Gly Val Asp Ser Ser Pro Arg 530 535 540 Ala Ala GluPro Val Gln Leu Asn Ser Cys Leu Ala Ala Gly Asp Ile 545 550 555 560 LeuAla Leu Val Phe Gly Leu Leu Phe Ala Val Thr Ser Val Ala Phe 565 570 575Leu Val Gln Met Arg Arg Gln His Arg Arg Gly Thr Lys Gly Gly Val 580 585590 Ser Tyr Arg Pro Ala Glu Val Ala Glu Thr Gly Ala His His His His 595600 605 His His 610 2 1833 DNA Artificial Sequence Nucleic acid encodingG250-GM-CSF fusion protein with His tag 2 atgtggctgc agagcctgctgctcttgggc actgtggcct gcagcatctc tgcacccgcc 60 cgctcgccca gccccagcacgcagccctgg gagcatgtga atgccatcca ggaggcccgg 120 cgtctcctga acctgagtagagacactgct gctgagatga atgaaacagt agaagtcatc 180 tcagaaatgt ttgacctccaggagccgacc tgcctacaga cccgcctgga gctgtacaag 240 cagggcctgc ggggcagcctcaccaagctc aagggcccct tgaccatgat ggccagccac 300 tacaagcagc actgccctccaaccccggaa acttcctgtg caacccagat tatcaccttt 360 gaaagtttca aagagaacctgaaggacttt ctgcttgtca tcccctttga ctgctgggag 420 ccagtccagg aggcggccgctcccctgtgc cccagcccct ggctccctct gttgatcccg 480 gcccctgctc caggcctcactgtgcaactg ctgctgtcac tgctgcttct gatgcctgtc 540 catccccaga ggttgccccggatgcaggag gattccccct tgggaggagg ctcttctggg 600 gaagatgacc cactgggcgaggaggatctg cccagtgaag aggattcacc cagagaggag 660 gatccacccg gagaggaggatctacctgga gaggaggatc tacctggaga ggaggatcta 720 cctgaagtta agcctaaatcagaagaagag ggctccctga agttagagga tctacctact 780 gttgaggctc ctggagatcctcaagaaccc cagaataatg cccacaggga caaagaaggg 840 gatgaccaga gtcattggcgctatggaggc gacccgccct ggccccgggt gtccccagcc 900 tgcgcgggcc gcttccagtccccggtggat atccgccccc agctcgccgc cttctgcccg 960 gccctgcgcc ccctggaactcctgggcttc cagctcccgc cgctcccaga actgcgcctg 1020 cgcaacaatg gccacagtgtgcaactgacc ctgcctcctg ggctagagat ggctctgggt 1080 cccgggcggg agtaccgggctctgcagctg catctgcact ggggggctgc aggtcgtccg 1140 ggctcggagc acactgtggaaggccaccgt ttccctgccg agatccacgt ggttcacctc 1200 agcaccgcct ttgccagagttgacgaggcc ttggggcgcc cgggaggcct ggccgtgttg 1260 gccgcctttc tggaggagggcccggaagaa aacagtgcct atgagcagtt gctgtctcgc 1320 ttggaagaaa tcgctgaggaaggctcagag actcaggtcc caggactgga catatctgca 1380 ctcctgccct ctgacttcagccgctacttc caatatgagg ggtctctgac tacaccgccc 1440 tgtgcccagg gtgtcatctggactgtgttt aaccagacag tgatgctgag tgctaagcag 1500 ctccacaccc tctctgacaccctgtgggga cctggtgact ctcggctaca gctgaacttc 1560 cgagcgacgc agcctttgaatgggcgagtg attgaggcct ccttccctgc tggagtggac 1620 agcagtcctc gggctgctgagccagtccag ctgaattcct gcctggctgc tggtgacatc 1680 ctagccctgg tttttggcctcctttttgct gtcaccagcg tcgcgttcct tgtgcagatg 1740 agaaggcagc acagaaggggaaccaaaggg ggtgtgagct accgcccagc agaggtagcc 1800 gagactggag cctagatggtgatggtgatg gtg 1833 3 11 PRT Artificial Sequence linker 3 Gly Gly GlyGly Ser Gly Gly Gly Gly Gly Ser 1 5 10 4 12 PRT Artificial Sequencelinker 4 Ala Gly Ser Ala Gly Ser Ala Gly Ser Ala Gly Ser 1 5 10 5 25 DNAArtificial Sequence primer 5 gcgggaattc atgtggctgc agagc 25 6 30 DNAArtificial Sequence primer 6 gagggaggcg gccgcctcct ggactggctc 30 7 27DNA Artificial Sequence primer 7 gagggagcgg ccgctcccct gtgcccc 27 8 58DNA Artificial Sequence primer 8 gcagaggtag agatctctaa tggtgatggtgatggtgggc tccagtctcg gctacctc 58 9 59 DNA Artificial Sequence primer 9ggagagatct ctaatgatga tgatgatgat gatgatgggc tccagtctcg gctacctct 59 1025 DNA Artificial Sequence primer 10 gagggcggcc gctcccctgt gcccc 25 1122 DNA Artificial Sequence primer 11 caactccatc atgaagtgtg ac 22 12 21DNA Artificial Sequence primer 12 ccacacggag tacttgcgct c 21 13 20 DNAArtificial Sequence primer 13 ccatgatggc cagccactac 20 14 21 DNAArtificial Sequence primer 14 cttgtttcat gagagagcag c 21 15 20 DNAArtificial Sequence primer 15 tctcgaaccc cgagtgacaa 20 16 21 DNAArtificial Sequence primer 16 tacgacggca aggattacat c 21 17 29 DNAArtificial Sequence primer 17 atgaaatata caagttatat cttggcttt 29 18 25DNA Artificial Sequence primer 18 atgctcttcg acctcgaaac agcat 25 19 24DNA Artificial Sequence primer 19 ggaattaata attacaagaa tccc 24 20 24DNA Artificial Sequence primer 20 gtttcagatc ccctttagtt ccag 24 21 20DNA Artificial Sequence primer 21 cttccccctc tgttcttcct 20 22 20 DNAArtificial Sequence primer 22 ttcctgtcga gccgtttcag 20

What is claimed is:
 1. A construct comprising a G250 kidney cancerspecific antigen attached to a granulocyte macrophage colony stimulatingfactor (GM-CSF).
 2. The construct of claim 1, wherein said GM-CSF is ahuman GM-CSF.
 3. The construct of claim 1, wherein the G250 antigen is ahuman G250 antigen.
 4. The construct of claim 1, wherein the G250antigen is covalently coupled to the GM-CSF.
 5. The construct of claim4, wherein the G250 antigen is coupled to the GM-CSF by a linker encodedby the nucleotide sequence gcggcg.
 6. The construct of claim 1, whereinthe G250 antigen and the GM-CSF are components of a fusion protein. 7.The construct of claim 6, wherein the G250 antigen and the GM-CSF arejoined by a peptide linker ranging in length from 2 to about 20 aminoacids.
 8. The construct of claim 7, wherein said peptide linker is-Arg-Arg-.
 9. The construct of claim 7, wherein said construct has thesequence of SEQ ID NO:
 1. 10. A composition comprising a G250 kidneycancer specific antigen attached to a granulocyte macrophage colonystimulating factor (GM-CSF), and a pharmaceutically acceptable diluentor excipient.
 11. The composition of claim 10, wherein said GM-CSF is ahuman GM-CSF and said G250 antigen is a human G250 antigen.
 12. Thecomposition of claim 10, wherein the G250 antigen is covalently coupledto the GM-CSF.
 13. The composition of claim 10, wherein the G250 antigenand the GM-CSF are components of a fusion protein.
 14. The compositionof claim 13, wherein the G250 antigen and the GM-CSF are joined by apeptide linker ranging in length from 2 to about 20 amino acids.
 15. Thecomposition of claim 14, wherein said peptide linker is -Arg-Arg-. 16.The composition of claim 10, further comprising an adjuvant.
 17. Thecomposition of claim 14, wherein said fusion protein has the sequence ofSEQ ID NO:
 1. 18. A nucleic acid encoding a fusion protein comprising aG250 kidney cancer specific antigen attached to a granulocyte macrophagecolony stimulating factor (GM-CSF).
 19. The nucleic acid of claim 18,wherein said nucleic acid is a deoxyribonucleic acid (DNA).
 20. Thenucleic acid of claim 18, wherein the G250 antigen is a human G250antigen and the GM-CSF is a human GM-CSF.
 21. The nucleic acid of claim20, wherein the G250 antigen and the GM-CSF are joined by a peptidelinker ranging in length from 2 to about 20 amino acids.
 22. The nucleicacid of claim 21, wherein said peptide linker is -Arg-Arg-.
 23. Thenucleic acid of claim 22, wherein said nucleic acid encodes thepolypeptide of SEQ ID NO:
 1. 24. The nucleic acid of claim 22, whereinsaid nucleic acid comprises the nucleic acid of SEQ ID NO:
 2. 25. Thenucleic acid of claim 20, wherein said nucleic acid is present in anexpression cassette.
 26. The nucleic acid of claim 20, wherein saidnucleic acid is present in a vector.
 27. The nucleic acid of claim 26,wherein said vector is a baculoviral vector.
 28. A host cell transfectedwith a nucleic acid comprising the nucleic acid of claim
 18. 29. Thehost cell of claim 28, wherein said host cell is a eukaryotic cell. 30.The host cell of claim 28, wherein said host cell is an insect cell. 31.A method of producing an anti-tumor vaccine, said method comprising:culturing a cell transfected with a nucleic acid comprising the nucleicacid of claim 18 under conditions where said nucleic expresses aG250-GM-CSF fusion protein; and recovering said fusion protein.
 32. Themethod of claim 31, wherein said cell is an insect cell.
 33. A method ofinducing an immune response against the G250 kidney-specific antigen ora cell displaying the G250 kidney-specific antigen, said methodcomprising: activating a cell of the immune system with a constructcomprising a kidney cancer specific antigen (G250) attached to agranulocyte macrophage colony stimulating factor (GM-CSF) whereby saidactivating provides an immune response directed against the G250antigen.
 34. The method of claim 33, wherein said activating comprisescontacting an antigen presenting cell with said construct.
 35. Themethod of claim 34, wherein said antigen presenting cell is a dendriticcell.
 36. The method of claim 35, wherein said antigen presenting cellis ex vivo.
 37. The method of claim 57, wherein the activated cell is acytotoxic T-lymphocyte (CTL).
 38. The method of claim 33, wherein saidactivating comprises systemic administration of said construct.
 39. Themethod of claim 38, wherein said activating comprises injecting saidconstruct into a mammal.
 40. The method of claim 39, wherein said mammalis a mammal selected from the group consisting of a human, a non-humanprimate, a rodent, a porcine, a largomorph, a canine, a feline, anequine, a porcine, and a bovine.
 41. The method of claim 40, whereinsaid mammal is a human diagnosed as having a renal cell carcinoma or acervical cancer.
 42. The method of claim 33, wherein said activatingcomprises contacting a cell selected from the group consisting of aperipheral blood lymphocyte (PBL), a dendritic cell, and a tumorinfiltrating lymphocyte (TIL), with said construct.
 43. The method ofclaim 42, wherein said contacting comprises contacting blood cells of amammal with said constuct ex vivo.
 44. The method of claim 43, furthercomprising re-infusing CTLs back into said mammal.
 45. The method ofclaim 33, wherein said activating comprises loading an antigenpresenting cell (APC) with a polypeptide comprising a GM-CSF-G250 fusionprotein.
 46. The method of claim 33, wherein said activating comprisesloading a dendritic cell (DC) with a polypeptide comprising aGM-CSF-G250 fusion protein.
 47. The method of claim 33, wherein saidactivating comprises introducing a nucleic acid encoding a GM-CSF-G250fusion protein into a mammal.
 48. The method of claim 47, wherein saidactivating comprises introducing a nucleic acid encoding a GM-CSF-G250fusion protein into muscle tissue in said mammal.
 49. The method ofclaim 47, wherein said activating comprises injecting a nucleic acidencoding a GM-CSF-G250 fusion protein into a mammal.
 50. The method ofclaim 33, wherein said method further comprises contacting said cellwith an immunomodulatory cytokine or drug.
 51. The method of claim 33,wherein said activating comprises transfecting a cell with a nucleicacid encoding a GM-CSF-G250 fusion protein.
 52. The method of claim 51,wherein said cell is selected from the group consisting of a dendriticcell (DC), a peripheral blood lymphcyte (PBL), an antigen presentingcell (APC), a tumor infiltrating lymphocyte (TIL), a fibroblast, acervical cancer cell, and a renal cell carcinoma tumor cell.
 53. Themethod of claim 51, wherein said transfecting is by use of an agent thattransfects a cell, said agent selected from the group consisting of aviral vector, a lipid, a liposome, a dendrimer, and a cationic lipid.54. A method of inhibiting the proliferation or growth of a transformedkidney cell, said method comprising: activating a cell of the immunesystem with a construct comprising a kidney cancer specific antigen(G250) attached to a granulocyte macrophage colony stimulating factor(GM-CSF) whereby said activating provides an immune response directedagainst the G250 antigen and said immune response inhibits the growth orproliferation of a transformed kidney cell.
 55. The method of claim 54,herein said transformed kidney cell is a renal cell carcinoma cell. 56.The method of claim 54, herein said transformed kidney cell is ametastatic cell.
 57. The method of claim 54, wherein said activatingcomprises contacting an antigen presenting cell with said construct. 58.The method of claim 57, wherein said antigen presenting cell is adendritic cell.
 59. The method of claim 57, wherein the activated cellis a cytotoxic T-lymphocyte (CTL).
 60. The method of claim 54, whereinsaid activating comprises systemic administration of said construct. 61.The method of claim 54, wherein said activating comprises injecting amammal with a DNA comprising an expression cassette comprising a nucleicacid encoding said construct.
 62. The method of claim 38, wherein saidactivating comprises injecting said construct into a mammal.
 63. Themethod of claim 62, wherein said mammal is a mammal selected from thegroup consisting of a human, a non-human primate, a rodent, a porcine, alargomorph, a canine, a feline, an equine, a porcine, and a bovine. 64.The method of claim 62, wherein said mammal is a human diagnosed ashaving a renal cell carcinoma or a cervical cancer.
 65. The method ofclaim 54, wherein said activating comprises contacting a cell selectedfrom the group consisting of a peripheral blood lymphocyte (PBL), adendritic cell, and a tumor infiltrating lymphocyte (TIL), with saidconstruct.
 66. The method of claim 65, wherein said contacting comprisescontacting blood cells of a mammal with said constuct ex vivo.
 67. Themethod of claim 66 further comprising re-infusing CTLs back into saidmammal.
 68. The method of claim 54, wherein said activating comprisesloading an antigen presenting cell (APC) with a polypeptide comprising aGM-CSF-G250 fusion protein.
 69. The method of claim 54, wherein saidactivating comprises loading a dendritic cell (DC) with a polypeptidecomprising a GM-CSF-G250 fusion protein.
 70. The method of claim 54,wherein said activating comprises introducing a nucleic acid encoding aGM-CSF-G250 fusion protein into a mammal.
 71. The method of claim 70,wherein said activating comprises introducing a nucleic acid encoding aGM-CSF-G250 fusion protein into muscle tissue in said mammal.
 72. Themethod of claim 54, wherein said activating comprises injecting anucleic acid encoding a GM-CSF-G250 fusion protein into a mammal. 73.The method of claim 54, wherein said method further comprises contactingsaid cell with an immunomodulatory cytokine or drug.
 74. The method ofclaim 54, wherein said activating comprises transfecting a cell with anucleic acid encoding a GM-CSF-G250 fusion protein.
 75. The method ofclaim 74, wherein said cell is selected from the group consisting of adendritic cell (DC), a peripheral blood lymphcyte (PBL), an antigenpresenting cell (APC), a tumor infiltrating lymphocyte (TIL), afibroblast, a cervical cancer cell, and a renal cell carcinoma tumorcell.
 76. The method of claim 74, wherein said transfecting is by use ofan agent that transfects a cell, said agent selected from the groupconsisting of a viral vector, a lipid, a liposome, a dendrimer, and acationic lipid.
 77. The method of claim 54, wherein said method furthercomprises contacting said cell with an immunomodulatory cytokine ordrug.
 78. A method of inhibiting the proliferation or growth of atransformed renal cell that bears a G250 antigen, said methodcomprising: removing an immune cell from a mammalian host; activatingsaid immune cell by contacting said cell with a protein comprising arenal cell carcinoma specific antigen (G250) attached to a granulocytemacrophage colony stimulating factor (GM-CSF) or a fragment thereof;optionally expanding the activated cell; and infusing the activated cellinto an organism containing a transformed renal cell bearing a G250antigen.
 79. The method of claim 78, wherein said removing comprisesobtaining peripheral blood lymphocytes or TILs from said mammalian host.80. The method of claim 79, wherein said infusing comprises infusing theactivated cells into the host from which the immune cell was removed.81. The method of claim 79, wherein said immune cell is selected fromthe group consisting of a dendritic cell, an antigen presenting cell, aB lymphocyte, a T-cell, and a tumor infiltrating lymphocyte.
 82. Amethod of treating an individual having a renal cell cancer, said methodcomprising: (a) sensitizing antigen presenting cells in vitro with asensitizing-effective amount of a chimeric fusion protein comprising arenal cell carcinoma specific antigen (G250) attached to a granulocytemacrophage colony stimulating factor (GM-CSF); and (b) administering toan individual having said renal cell cancer or metastasis atherapeutically effective amount of the sensitized antigen presentingcells.
 83. The method of claim 82, wherein the antigen presenting cellsare autologous to the individual or are MHC matched allogenic dendriticcells.
 84. The method of claim 82, wherein said sensitizing comprisescontacting cells selected from the group consisting of peripheral bloodlymphocytes, monocytes, fibroblasts, TILs, and dendritic cells with saidchimeric fusion protein.
 85. The method of claim 82, wherein saidsensitizing comprises transfecting dendritic cells or RCCs with anucleic acid encoding said chimeric fusion protein.